Assays for monitoring cell motility in real-time

ABSTRACT

The invention relates to devices, devices for arraying biomolecules, including cells, methods for arraying biomolecules, assays for monitoring cellular movement, and systems for monitoring cellular movement. The devices include a support; a first layer configured to be placed in fluid-tight contact with the support, the first layer having an upper surface and defining a pattern of micro-orifices, each micro-orifice of the pattern of micro-orifices having walls and defining a micro-region on the support when the first layer is placed in fluid-tight contact with the support such that the walls of said each micro-orifice and the micro-region on the support together define a micro-well; and a second layer configured to be placed in fluid-tight contact with the upper surface of the first layer, the second layer defining a pattern of macro-orifices, each macro-orifice of the pattern of macro-orifices having walls and defining a macro-region when the first layer is placed in fluid-tight contact with the support and the second layer is placed in fluid-tight contact with the first layer such that the walls of the macro-orifice and the macro-region together define a macro-well.

RELATED APPLICATIONS

[0001] This application claims the benefit of and incorporates herein byreference, in their entirety: U.S. application Ser. No. 09/709,776,filed on Nov. 8, 2000; U.S. Provisional Application No. 60/307,886,filed on Jul. 27, 2001; U.S. Provisional Application No. 60/323,742,filed on Sep. 21, 2001; U.S. Provisional Application No. 60/328,103,filed on Oct. 11, 2001; U.S. Provisional Application No. 60/330,456,filed on Oct. 22, 2001; U.S. Provisional Application No. 60/334,548,filed on Dec. 3, 2001; U.S. Provisional Application No. 60/363,355,filed on Mar. 12, 2002; and U.S. Provisional Application No. 60/374,799,filed on Apr. 24, 2002.

FIELD OF THE INVENTION

[0002] The invention generally relates to devices and methods forpatterning cells in a predetermined array for subsequent observation andmeasurement of cell motility.

BACKGROUND

[0003] The study of cellular behavior and the effects of externalstimuli on the cell are prevalent throughout contemporary biologicalresearch. Generally, this research involves exposing a cell to externalstimuli and studying the cell's reaction. By placing a living cell intovarious environments and exposing it to different external stimuli, boththe internal workings of the cell and the effects of the externalstimuli on the cell can be measured, recorded, and better understood.

[0004] When a cell is exposed to chemical stimuli, its behavior is animportant consideration, particularly when developing and evaluatingtherapeutic candidates and their effectiveness. By documenting thereaction of a cell or a group of cells to a chemical stimulus, such as atherapeutic agent, the effectiveness of the chemical stimulus can bebetter understood. In particular, in the fields of oncology and cellbiology, cell migration and metastasis are regularly considered.Typically, studies in these fields involve analyzing the migration andbehavior of living cells with regard to various biological factors andpotential anti-cancer drugs. Moreover, the resultant migration,differentiation, and behavior of a cell are often insightful towardsfurther understanding the chemotactic processes involved in tumor cellmetastasis. In addition, these studies can also provide insight into theprocesses of tissue regeneration, wound healing, inflamation, autoimmunediseases, and many other degenerative diseases and conditions.

[0005] Cell migration assays are often used in conducting these types ofresearch. Commercially available devices for creating such assays areoften based on or employ a Boyden chamber (a vessel partitioned by athin porous membrane to form two distinct, super-imposed chambers). Alsoknown as transwells, the Boyden chamber is used by placing a migratorystimulus on one side of a thin porous membrane and cells to be studiedon the other. After a sufficient incubation period the cells may befixed, stained, and counted to study the effects of the stimulus on cellmigration across the membrane.

[0006] The use of transwells has several shortcomings. For instance,assays employing transwells require a labor-intensive protocol that isnot readily adaptable to high-throughput screening and processing. Thecounting of cells, which is often done manually using a microscope, is atime-consuming, tedious, and expensive process. Furthermore, cellcounting is also subjective and involves statistical approximations.Specifically, due to the time and expense associated with examining anentire filter, only representative areas, selected at random, may becounted, and, even when these areas are counted, if a cell has onlypartially migrated through the filter, a technician must, nevertheless,exercise his or her judgement when accounting for such a cell.

[0007] Notwithstanding the above, perhaps the most significantdisadvantage to the use of transwells is that when the cells are fixedto a slide, as required for observation, they are killed. Consequently,once a cell is observed it can no longer be reintroduced into the assayor studied at subsequent periods of exposure to the stimulus. Therefore,in order to study the progress of a cell reaction to a stimulus, it isnecessary to run concurrent samples that may be slated for observationat various time periods before and after the introduction of thestimulus. In light of the multiple samples required for each test, inaddition to the positive and negative controls required to obtainreliable data, a single chemotaxis assay can require dozens of filters,each of which needs to be individually examined and counted—an enormousand onerous task.

[0008] Cell migration and differentiation is also important to theunderstanding of numerous biological functions, both normal andabnormal. For example, the study of tissue regeneration and woundhealing, and the study of inflamation, autoimmune diseases and otherdegenerative diseases, all involve the analysis of cell movement, eitherspontaneous or in response to chemotactic factors, or other cellularsignals. Further, in studying the treatment of various abnormal cellularfunctions or diseases, scientists must analyze the effects of potentialtherapies on cell movement in cell culture before proceeding to clinicalstudies.

[0009] Thus, a cell migration assay is a useful tool for cell biologistsfor determining the ability of cells to grow, proliferate, and migrate.Although useful, assays based on cell migration have been limited in usebecause of the unavailability of convenient tools for performing theassay. Currently, commercially available devices for studying cellmigration or chemotaxis are based on the Boyden Chamber. S. Boyden, J.Exp. Med. 115: pp. 453-466, (1962). Also known as transwells, thesedevices are used generally as follows: a migratory stimulus is added toone side of a thin porous membrane; cells are then added to the otherside, and the device is incubated. After a given time, cells that havenot migrated across the membrane are removed, and the cells that havemigrated are counted, usually after fixing and staining.

[0010] There are several disadvantages to this procedure. The use of aBoyden Chamber requires a labor-intensive protocol, and it is notreadily adaptable to a high-throughput screening process. Theexamination and counting of the cells on the filter is time-consuming,tedious, and expensive. It is also highly subjective because itnecessarily involves the exercise of judgment in determining whether tocount a cell that has only partially migrated across the filter. Inaddition, the time and expense associated with examining the entirefilter necessitates that only representative areas, selected at random,be counted, thus rendering the results less accurate than wouldotherwise be the case if the entire filter were examined and counted.

[0011] Perhaps the most important disadvantage in this procedure is thatthe fixing step kills the cells. That is, the procedure is destructiveof the cell sample. Thus, in order to determine a time-dependentrelationship of the chemotactic response; that is, a kinetic study, of aparticular chemical agent, it is necessary to run multiple samples foreach of multiple time periods. When one considers that multiple samples,as well as positive and negative controls, are necessary to obtainreliable data, a single chemotaxis assay can produce dozens of filters,each of which needs to be individually examined and counted. The timeand expense associated with a time-dependent study is usuallyprohibitive of conducting such a study using the Boyden procedure. Asthe migratory behavior of cells has potential implications in thedevelopment of certain therapeutics, a better in vitro system is neededfor screening and quantifying the effects of drug targets on cellmotility and migration.

[0012] Alternatives to the Boyden assay have been proposed to overcomesome of the above disadvantages. See generally, P. Wilkinson, Methods inEnzymology, Vol. 162, (Academic Press, Inc. 1988), pp. 38-50; see also,Goodwin, U.S. Pat. No. 5,302,515; Guiruis et al., U.S. Pat. No.4,912,057; Goodwin, U.S. Pat. No. 5,284,753; and Goodwin, U.S. Pat. No.5,210,021. Although the chemotaxis devices and procedures described inthese references have some advantages over the original Boyden procedureand apparatus, they are not without their shortcomings. For example, allof these procedures, like the Boyden Chamber, require that the filter beremoved and the non-migrated cells be wiped or brushed from the filterbefore the migrated cells can be counted. In addition, most of theseprocedures require fixing and staining the cells, and none of thempermit the kinetic or time-dependent study of the chemotactic responseof the same cell sample. Further, these methods involve the counting ofcells, a lengthy procedure not compatible with high-throughputapplications.

[0013] Cell migration is important for tissue morphogenesis. Muchprogress has been made in terms of understanding the molecular basis ofcell movement. However, because of the inherent complexity ofmulticellular systems, little is known about how cell migration mediatescellular pattern formation. Bragwynne et al. (Proceedings of the 22ndAnnual International Conference, Jul. 23-28, 2000) report spontaneouspattern generation in a model mammalian tissue in vitro by spatiallyconstraining cell adhesion. They observed coupled, coordinated migrationof bovine capillary endothelial cells within a field defined by spatiallimits of an adhesive surface. Bragwynne et al. have speculated thatpattern-generating behavior that emerges from collective interactionsamong different interacting cellular components may contribute to tissuedevelopment. Bragwynne et al. surmise that the resulting cell patternsdemonstrate that a geometric constraint on a group of migratory cellscan induce spontaneous pattern formation. Thus, in order to more fullyunderstand spontaneous pattern formation it is necessary to have adevice that would allow one to pattern cells in a predetermined locationin a predefined pattern and observe their migration and spontaneouspattern formation.

[0014] The role of cell-cell interactions in the control of cellulargrowth, migration, differentiation, and function is becomingincreasingly apparent. Cell-cell contact is believed to be involved indevelopmental process such as mesoderm interaction andmesenchymal-epithelial transformation. Sargent, T. D., et al., Dev.Biol. 114:238-246 (1986); Lehtonen, E., et al., J. Embryol. Exp.Morphol. 33:187-203 (1975). In the nervous system, the pattern of neuralcell migration axonal cone growth and glial cell differentiation arethought to depend on heterolytic cell-cell interactions. Rakic, P., Thecell in contact, New York: Wiley Intersciences, 67-91 (1985); Bently,D., et al., Nature 304:62-65 (1983); Lillien, L., et al., Neuron4:525-534 (1990). In the immune system, the development and activationof lymphocytes are dependent on contact with a number of different celltypes throughout the life of a lymphocyte. Kierny, P.C., et al., Blood70:1418-1424 (1987). In addition, the differentiation and function ofepithelial cells, e.g. intestinal epithelia, are regulated in part bycontacts with the underlying mesenchymal cells. Kedinger, M., et al.,Cell Differ. 20:171-182 (1987). As the role of heterocellular contactbecomes more apparent, in vitro systems designed to investigateintercellular communication are needed.

[0015] A number of experimental approaches utilizing co-cultures of twodifferent tissue or cell types have been used to examine the role ofintercellular communication in various cellular processes. For example,the contribution of cell-cell interactions to embryonic inductiveprocesses was elucidated by experiments in which pieces of embryonictissue were attached to opposite sides of a porous membrane. Grobstein,C., Exp. Cell Res. 10:424-440 (1956). Investigations of the effects ofheterotypic interactions on cellular functions have co-cultured twodifferent cell types in the same culture dish. Davies, P. F., et al., J.Cell Biol. 101:871-879 (1985); Guguen-Guillouzo, C., et al., Exp. CellRes. 143:47-54 (1983); Mehta, R. P., et al., Cell 44:187-196 (1986);Orlidge, A., et al., J. Cell Biol. 105:1455-1462 (1987); Shimaoka, S.,et al., Exp. Cell Res. 172:228-242 (1987). These co-cultures havelimited use, however, because they represent a mixed population ofcells. The effects of intercellular contact on cell morphology or on afunction or protein unique to one of the cell types can be examined;however, investigation of biochemical or molecular processes common toboth cells in not possible. Porous filters have been used in co-culturesof tissue culture cells to circumvent this limitation. In these studies,one cell type is usually grown in a tissue culture dish and second celltype cultured on a porous membrane in a chamber that fits into theculture dish. Hisanaga, K., et al., Dev. Brain Res. 54:151-160 (1990);Kruegar, G. G., et al., Dermatologic 179:91S-100S (1989); Ueda, H., etal., J. Cell Sci. 89:175-188 (1988).

[0016] It has been determined that many factors operate synergisticallyto produce an effect on cellular migration. For example, Woodward etal., Journal of Cell Science 111, 469-478 (1998) have used a migrationchamber to demonstrate that α_(v)β₃ integrin and PDGF receptor worksynergistically to increase cell migration. Thus, an assay device ormethod that would allow further study of cell migration in response tovarious factors, including synergistic effects, would aid in theunderstanding of cellular motility and migration.

[0017] To study cell motility, either in response to a cell affectingagent, or random motility, it is desirable to be able to monitorcellular movement from a predefined “starting” position. To do this,cells must be placed, attached or immobilized upon a surface in such amanner that their viability is maintained and that their position isdefineable so that multiple interrogations or probing of cellularresponse (i.e. motility or lack thereof) may be performed. In previousmethods concerning cell immobilization, cells often undergo anonreversible immobilization. For example, cells have been immobilizedby patterning cells on a self-assembled monolayer that has a proteintether that will “capture” the cell. Alternatively, cells have beenimmobilized via immunological reaction with antibodies, which themselveshave been immobilized on the immobilization surface. Other methods ofimmobilization involve simply allowing cells to attach themselves to asuitable surface, such as glass or plastic, and then allowing them tomigrate into adjacent areas.

[0018] Ostuni et al. have used elastomeric membranes to pattern theattachment of cells to surfaces that are commonly used in cell culture.Patterning of cells is an experimental protocol that is broadly usefulin studying and controlling the behavior of anchorage-dependent cells.Chen, C. S., et al., Science, 276, 1425-1428 (1997); Ingber, D. E., etal., J. Cell Biol. 109, 317-330 (1989); Ingber, D. E. Proc. Natl. Acad.Sci. U.S.A., 87, 3379-3583 (1990); Singhvi, R.; et al., Science 264,696-698 (1994). It is also relevant to applied cell biology,bio-sensors, high-throughput screening and tissue engineering. Chen, etal., Science 276, 1425-1428 (1997); Bhatia, S. N. et al., Biotechnol. J.14, 378-387 (1998); Borkholder, D. A., et al., J. Neurosci. Methods, 77,61-66 (1997); Dodd, S. J., et al., Biophys. J, 76, 103-109 (1999);Fromherz, P., Phys. Rev. Lett. 78, 4131-4134; Hickman, J. J., et al., J.Vac. Sci. Technol., A-Vac. Surf. Films 12, 607-616 (1994); Humes, H. D.,et al., Nat. Biotechnol. 17, 451-455 (1999); Huynh, T., et al., Nat.Biotechnol. 17, 1088-1086 (1999); Kapur, R., et al., J. Biomech.Eng-Trans. ASME 121, 65-72 (1999); Pancrazio, J. J., et al., Sens.Actuators, B-Chem. 53, 179-185 (1998); St. John, P. M., et al., Anal.Chem. 70, 1108-1111 (1998); You, A. J., et al., Chem., Biol. 4, 969-975(1997).

[0019] Soft lithography has been developed to provide a set of methodsfor patterning surfaces and fabricating structures with dimensions inthe 1-100 μm range in ways that are useful in cell biology andbiochemistry. Qin, D., et al., Adv. Mater. 8, 917-919 (1996); Qin, D.,et al., J. Vac. Sci., Technol., B 16, 98-103 (1998); Xia, Y., et al.,Agnew. Chem., Int. Ed. Engl. 37, 550-575 (1998); Zhao, X. -M., et al.,Adv. Mater. 8, 837-840 (1996); Zhao, X. -M., et al., Adv. Mater. 9,251-254 (1997). Microcontact printing is particularly useful as a methodfor generating patterns of proteins and cells, by patterningself-assembled monolayers of alkanethiolates on the surface of gold.Chen, C. S., et al., Science 276, 1425-1428 (1997); Singhvi, R., et al.,Science 264, 696-698 (1994); López, G. P., et al., J. Am. Chem. Soc.115, 5877-5878 (1993); Kumar, A., et al., Appl. Phys. Lett. 63,2002-2004 (1993); Mrksich, M., et al., Trends Biotech. 13, 228-235(1995).

[0020] Mrksich et al. have partitioned a gold support into regionspatterned with a hydrophobic alkanethiolate and another alkanethiolatethat presents small percentages of an electrochemically active terminalgroup. (Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Submitted.). Aftercells attached and spread themselves on the hydrophobic pattern,application of a short voltage pulse changed the oxidation state andpolarity of the terminal redox center. This oxidation state and polaritychange allowed groups presenting peptide sequences to react with thesurface to generate a subsequent surface that the patterned cells couldspread on. This method requires the synthesis of electroactivealkanethiols, and also requires electrochemical instrumentation.

[0021] It is further known in the art to use under agarose migrationstudies to assay cell differentiation and cell migration. These methodsare slow and laborious and as such are not suitable to the demands ofhigh throughput assays.

[0022] Thus, there remains a need for a device and method of trackinglive cells in real time. Current existing techniques require laboriousprotocols and work as end-point assays.

SUMMARY OF THE INVENTION

[0023] The present invention provides a device comprising a support; afirst layer configured to be placed in fluid-tight contact with thesupport, the first layer having an upper surface and defining a patternof micro-orifices, each micro-orifice of the pattern of micro-orificeshaving walls and defining a micro-region on the support when the firstlayer is placed in fluid-tight contact with the support such that thewalls of said each micro-orifice and the micro-region on the supporttogether define a micro-well; and a second layer configured to be placedin fluid-tight contact with the upper surface of the first layer, thesecond layer defining a pattern of macro-orifices, each macro-orifice ofthe pattern of macro-orifices having walls and defining a macro-regionwhen the first layer is placed in fluid-tight contact with the supportand the second layer is placed in fluid-tight contact with the firstlayer such that the walls of the macro-orifice and the macro-regiontogether define a macro-well.

[0024] The first layer is preferably configured to be placed inconformal contact with the support when the first layer is placedagainst the support. The second layer is preferably configured to beplaced in conformal contact with the first layer when the second layeris placed against the first layer. The support is made of a materialselected from the group consisting of glass, silicon, fused silica,metal films, polystyrene, poly(methylacrylate) and polycarbonate. Thefirst layer and the second layer are made of a material selected fromthe group consisting of glass, elastomers, rigid plastics, metals,silicon and silicon dioxide. Preferably the first layer and second layerare made of an elastomer. Most preferably the first layer and the secondlayer is made of PDMS.

[0025] Preferably each macro-region encompasses at least onemicro-region and more preferably each macro-region encompasses aplurality of micro-regions.

[0026] In one embodiment the walls of each macro-well define a curve ina cross-sectional plane perpendicular to the upper surface of the firstlayer.

[0027] In the device at least one of the pattern of micro-orifices andthe pattern of macro-orifices spatially and dimensionally corresponds toa standard microtiter plate. Preferably the at least one of the patternof micro-orifices and the pattern of macro-orifices spatially anddimensionally corresponds to a standard microtiter plate selected from agroup consisting of a 6-well microtiter plate, a 12-well microtiterplate, a 24-well microtiter plate, a 96-well microtiter plate, a384-well microtiter plate, a 1,536-well microtiter plate, and a9,600-well microtiter plate.

[0028] In another embodiment, the device further comprises at least onecap for enclosing at least one of the macro-wells. Preferably thedevices comprises a plurality of caps for enclosing each of themacro-wells.

[0029] The device may also comprise a means for aligning themicro-orifices with the macro-orifices. The means for aligning includesa guide mechanism on at least one of the support, the first layer andthe second layer. The guide mechanism includes protrusions extendingfrom the support, and guide orifices defined in the first layer and inthe second layer for receiving the protrusions therein thereby aligningrespective ones of the first layer and the second layer on the support.The means for aligning includes markings on at least one of the support,the first layer and the second layer.

[0030] In another embodiment, the device comprises a support; a firstlayer configured to be placed in fluid-tight contact with the support,the first layer having an upper surface and defining a pattern ofmicro-orifices, each micro-orifice of the pattern of micro-orificeshaving walls and defining a micro-region on the support when the firstlayer is placed in fluid-tight contact with the support such that thewalls of said each micro-orifice and the micro-region on the supporttogether define a micro-well; and a second layer configured to be placedin fluid-tight contact with the support upon the removal of the firstlayer from the support, the second layer defining a pattern ofmacro-orifices, each macro-orifice of the pattern of macro-orificeshaving walls and defining a macro-region when the first layer is placedin fluid-tight contact with the support and the second layer is placedin fluid-tight contact with the first layer such that the walls of themacro-orifice and the macro-region together define a macro-well.

[0031] The present invention further provides a device comprising asupport; a first layer configured to be placed in fluid-tight contactwith the support, the first layer having an upper surface and defining apattern of micro-orifices, each micro-orifice of the pattern ofmicro-orifices having walls and defining a micro-region on the supportwhen the first layer is placed in fluid-tight contact with the supportsuch that the walls of said each micro-orifice and the micro-region onthe support together define a micro-well; and a second layer configuredto be placed in fluid-tight contact with the support, the second layercomprising a plurality of rings, the rings defining a pattern ofrespective macro-orifices, each ring having walls and defining amacro-region when the second layer is placed in fluid-tight contact withthe support such that the walls of the ring and the macro-regiontogether define a macro-well.

[0032] The invention further comprises a device comprising a support; alayer configured to be placed in fluid-tight contact with the support,the layer defining a pattern of macro-orifices, each macro-orifice ofthe pattern of macro-orifices having walls and defining a macro-regionwhen the second layer is placed in fluid-tight contact with the supportsuch that the walls of the macro-orifice and the macro-region togetherdefine a macro-well; and a set of plugs, each of the plugs beingconfigured for being received in a respective macro-well, each of theplugs comprising a lower membrane adapted to be placed in fluid-tightcontact with the support when the layer is placed in fluid-tight contactwith the support and the plug is received in a corresponding macro-welldefined by the layer and the support, the lower membrane furtherdefining a pattern of micro-orifices, wherein each micro-orifice haswalls and defines a micro-region on the support when the plug is influid-tight contact with the support such that the walls of themicro-orifice and the micro-region together define a micro-well.

[0033] The present invention also provides a device for arrayingbiomolecules, including cells, comprising a support; a first layerconfigured to be placed in fluid-tight contact with the support, thefirst layer having an upper surface and defining a pattern ofmicro-orifices, each micro-orifice of the pattern of micro-orificeshaving walls and defining a micro-region on the support when the firstlayer is placed in fluid-tight contact with the support such that thewalls of said each micro-orifice and the micro-region on the supporttogether define a micro-well; a second layer configured to be placed influid-tight contact with the upper surface of the first layer, thesecond layer defining a pattern of macro-orifices, each macro-orifice ofthe pattern of macro-orifices having walls and defining a macro-regionwhen the first layer is placed in fluid-tight contact with the supportand the second layer is placed in fluid-tight contact with the firstlayer such that the walls of the macro-orifice and the macro-regiontogether define a macro-well; wherein the first layer and the secondlayer are configured for an arraying of biomolecules and/or cells on thesupport through the pattern of micro-orifices and the pattern ofmacro-orifices.

[0034] Preferably the first layer is configured to be placed inconformal contact with the support when the first layer is placedagainst the support and the second layer is configured to be placed inconformal contact with the support when the second layer is placedagainst the support. The support is made of a material selected from thegroup consisting of glass, silicon, fused silica, metal films,polystyrene, poly(methylacrylate) and polycarbonate. The first layer andthe second layer are made of a material selected from the groupconsisting of glass, elastomers, rigid plastics, metals, silicon andsilicon dioxide. The first layer and second layer is preferably made ofan elastomer, and more preferably PDMS.

[0035] In the device preferably each macro-region encompasses at leastone micro-region and more preferably each macro-region encompasses aplurality of micro-regions.

[0036] In the device, the walls of each macro-well may define a curve ina cross-sectional plane perpendicular to the upper surface of the firstlayer.

[0037] Preferably, the device has at least one of the pattern ofmicro-orifices and the pattern of macro-orifices spatially anddimensionally corresponds to a standard microtiter plate. Further, theat least one of the pattern of micro-orifices and the pattern ofmacro-orifices spatially and dimensionally corresponds to a standardmicrotiter plate selected from a group consisting of a 6-well microtiterplate, a 12-well microtiter plate, a 24-well microtiter plate, a 96-wellmicrotiter plate, a 384-well microtiter plate, a 1,536-well microtiterplate, and a 9,600-well microtiter plate.

[0038] In one embodiment, the device further comprises at least one capfor enclosing at least one of the macro-wells. Preferably the devicescomprises a plurality of caps for enclosing each of the macro-wells.

[0039] The device may also comprise a means for aligning themicro-orifices with the macro-orifices. The means for aligning includesa guide mechanism on at least one of the support, the first layer andthe second layer. The guide mechanism includes protrusions extendingfrom the support, and guide orifices defined in the first layer and inthe second layer for receiving the protrusions therein thereby aligningrespective ones of the first layer and the second layer on the support.The means for aligning includes markings on at least one of the support,the first layer and the second layer.

[0040] The support has an upper surface that may have a coating thereon.The coating comprises a material selected from the group consisting ofproteins, protein fragments, peptides, small molecules, lipid bilayers,metals and self-assembled monolayers.

[0041] The present invention further provides a device for arrayingbiomolecules and/or cells comprising a support; a first layer configuredto be placed in fluid-tight contact with the support, the first layerhaving an upper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well; a second layer configured to be placed in fluid-tightcontact with the support, the second layer defining a pattern ofmacro-orifices, each macro-orifice of the pattern of macro-orificeshaving walls and defining a macro-region when the second layer is placedin fluid-tight contact with the support such that the walls of themacro-orifice and the macro-region together define a macro-well; whereinthe first layer and the second layer are configured for an arraying ofbiomolecules and/or cells on the support through the pattern ofmicro-orifices and the pattern of macro-orifices.

[0042] The present invention also provides a device for arrayingbiomolecules and/or cells comprising a support; a first layer configuredto be placed in fluid-tight contact with the support, the first layerhaving an upper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well; a second layer configured to be placed in fluid-tightcontact with the support, the second layer comprising a plurality ofrings, the rings defining a pattern of respective macro-orifices, eachring having walls and defining a macro-region when the second layer isplaced in fluid-tight contact with the support such that the walls ofthe ring and the macro-region together define a macro-well; wherein thefirst layer and the second layer are configured for an arraying ofbiomolecules and/or cells on the support through the pattern ofmicro-orifices and the pattern of macro-orifices.

[0043] In another embodiment, a device for arraying biomolecules and/orcells comprises a support; a layer configured to be placed influid-tight contact with the support, the layer defining a pattern ofmacro-orifices, each macro-orifice of the pattern of macro-orificeshaving walls and defining a macro-region when the second layer is placedin fluid-tight contact with the support such that the walls of themacro-orifice and the macro-region together define a macro-well; a setof plugs, each of the plugs being configured for being received in arespective macro-well, each of the plugs comprising a lower membraneadapted to be placed in fluid-tight contact with the support when thelayer is placed in fluid-tight contact with the support and the plug isreceived in a corresponding macro-well defined by the layer and thesupport, the lower membrane further defining a pattern ofmicro-orifices, wherein each micro-orifice has walls and defines amicro-region on the support when the plug is in fluid-tight contact withthe support such that the walls of the micro-orifice and themicro-region together define a micro-well; wherein the first layer andthe second layer are configured for an arraying of biomolecules and/orcells on the support through the pattern of micro-orifices and thepattern of macro-orifices.

[0044] The present invention further provides a method for arrayingbiomolecules and/or cells comprising the steps of positioning a firstlayer to be in fluid-tight contact with a support, the first layerhaving an upper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well; positioning a second layer to be in fluid-tight contact withan upper surface of the first layer, the second layer defining a patternof macro-orifices, each macro-orifice of the pattern of macro-orificeshaving walls and defining a macro-region when the first layer is placedin fluid-tight contact with the support and the second layer is placedin fluid-tight contact with the first layer such that the walls of themacro-orifice and the macro-region together define a macro-well; andimmobilizing at least one biomolecule and/or cell of a plurality ofbiomolecules and/or cells in each respective micro-region on the supportso as to situate the at least one biomolecule and/or cell within acorresponding micro-well, the biomolecules and/or cells thereby beingarrayed on the support in a pattern that corresponds to the pattern ofthe micro-orifices.

[0045] In another embodiment, a coating is applied to an upper surfaceof the support. The coating may be cells, proteins, protein fragments,peptides, small molecules, lipid bilayers, metals and self-assembledmonolayers.

[0046] The present invention further provides a method for arrayingbiomolecules and/or cells comprising: positioning a first layer to be influid-tight contact with a support, the first layer having an uppersurface and defining a pattern of micro-orifices, each micro-orifice ofthe pattern of micro-orifices having walls and defining a micro-regionon the support when the first layer is placed in fluid-tight contactwith the support such that the walls of said each micro-orifice and themicro-region on the support together define a micro-well; immobilizingat least one biomolecule and/or cell of a plurality of biomoleculesand/or cells in each respective micro-region on the support so as tosituate the at least one biomolecule and/or cell within a correspondingmicro-well, the biomolecules and/or cells thereby being arrayed on thesupport in a pattern that corresponds to the pattern of themicro-orifices; removing the first layer from the support after the stepof immobilizing; and positioning a second layer to be in fluid-tightcontact with the support, the second layer defining a pattern ofmacro-orifices, each macro-orifice of the pattern of macro-orificeshaving walls and defining a macro-region when the second layer is placedin fluid-tight contact with the support such that the walls of themacro-orifice and the macro-region together define a macro-well.

[0047] In an alternate embodiment, the method comprises positioning afirst layer to be in fluid-tight contact with a support, the first layerhaving an upper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well; positioning a second layer to be in fluid-tight contact withthe support, the second layer comprising a plurality of rings, the ringsdefining a pattern of respective macro-orifices, each ring having wallsand defining a macro-region when the second layer is placed influid-tight contact with the support such that the walls of the ring andthe macro-region together define a macro-well; and immobilizing at leastone biomolecule and/or cell of a plurality of biomolecules and/or cellsin each respective micro-region on the support so as to situate the atleast one biomolecule and/or cell within a corresponding micro-well, thebiomolecules and/or cells thereby being arrayed on the support in apattern that corresponds to the pattern of the micro-orifices.

[0048] In yet another embodiment, a method of arraying biomoleculesand/or cells comprises positioning a layer to be in fluid-tight contactwith the support, the layer defining a pattern of macro-orifices, eachmacro-orifice of the pattern of macro-orifices having walls and defininga macro-region when the layer is placed in fluid-tight contact with thesupport such that the walls of the macro-orifice and the macro-regiontogether define a macro-well; inserting each plug of a set of plugs in arespective macro-well, each of the plugs comprising a lower membraneplaced in fluid-tight contact with the support when the layer is placedin fluid-tight contact with the support and the plug is received in acorresponding macro-well defined by the layer and the support, the lowermembrane further defining a pattern of micro-orifices, wherein eachmicro-orifice has walls and defines a micro-region on the support whenthe plug is in fluid-tight contact with the support such that the wallsof the micro-orifice and the micro-region together define a micro-well;and immobilizing a biomolecule and/or cell in at least one micro-regionon the support so as to be situated within the micro-well, such that thebiomolecule and/or cell is arrayed on the support in a pattern thatcorresponds to the first pattern of micro-orifices.

[0049] The present invention further provides a method of fabricating adevice comprising: providing a support; providing a first layerconfigured to be placed in fluid-tight contact with the support, thefirst layer having an upper surface and defining a pattern ofmicro-orifices, each micro-orifice of the pattern of micro-orificeshaving walls and defining a micro-region on the support when the firstlayer is placed in fluid-tight contact with the support such that thewalls of said each micro-orifice and the micro-region on the supporttogether define a micro-well; and providing a second layer configured tobe placed in fluid-tight contact with the upper surface of the firstlayer, the second layer defining a pattern of macro-orifices, eachmacro-orifice of the pattern of macro-orifices having walls and defininga macro-region when the first layer is placed in fluid-tight contactwith the support and the second layer is placed in fluid-tight contactwith the first layer such that the walls of the macro-orifice and themacro-region together define a macro-well.

[0050] In one embodiment, the method comprises: providing a mold;applying an elastomeric material in liquid form to a mold having apattern of micro-posts corresponding to the pattern of micro-orifices;curing the elastomeric material; and removing the cured elastomericmaterial from the mold. The application includes spin-coating theelastomeric material. In an alternate embodiment, an adhesive adapted tobe applied between the first layer and the second layer when the secondlayer is placed against the first layer is provided.

[0051] In yet another embodiment, a method of fabricating a devicecomprises: providing a first precursor layer (preferably an elastomerand more preferably PDMS); curing the first precursor layer to form afirst layer, the first layer having an upper surface and defining apattern of micro-orifices, each micro-orifice of the pattern ofmicro-orifices having walls and defining a micro-region in a planedefined by a lower surface of the first layer; placing a mold having apattern of macro-posts on an upper surface of the first layer; providinga second precursor layer on the upper surface of the first layer; curingthe second precursor layer to form a second layer, the second layerdefining a pattern of macro-orifices, each macro-orifice of the patternof macro-orifices having walls and defining a macro-region in a planedefined by a lower surface of the second layer.

[0052] Another embodiment further comprises placing the first layeragainst a support for establishing a fluid-tight contact of the firstlayer with the support, each micro-orifice of the pattern ofmicro-orifices having walls and defining the micro-region on the supportwhen the first layer is placed in fluid-tight contact with the supportsuch that the walls of said each micro-orifice and the micro-region onthe support together define a micro-well, and each macro-orifice of thepattern of macro-orifices having walls and defining the macro-regionsuch that the walls of said each macro-orifice and the macro-regiontogether define a macro-well.

[0053] In yet another embodiment wherein providing a second precursorlayer comprises providing the second precursor layer on the mold havingthe pattern of macro-posts such that a macro-orifice created by eachmacro-post encompasses at least one or more preferably a plurality ofmicro-regions.

[0054] In another embodiment, there is provided a method of fabricatinga device, comprising: providing a first mold having a pattern ofmicro-posts; providing a second mold having a pattern of macro-posts;placing the second mold on the first mold; applying an elastomericprecursor in liquid form to the first mold and to the second mold afterthe step of placing so as to fill spaces around the micro-posts and themacro-posts with the elastomeric precursor; curing the elastomericprecursor after the step of applying for providing an elastomericelement; separating the elastomeric element from the first mold and fromthe second mold, wherein the pattern of micro-posts and the pattern ofmacro-posts are configured such that the micro-posts form a pattern ofmicro-orifices in the elastomeric element, and the macro-posts define apattern of macro-orifices in the elastomeric element.

[0055] The present invention also provides for assays measuring cellmovement. One embodiment, comprises: positioning a first layer to be influid-tight contact with a support, the first layer having an uppersurface and defining a pattern of micro-orifices, each micro-orifice ofthe pattern of micro-orifices having walls and defining a micro-regionon the support when the first layer is placed in fluid-tight contactwith the support such that the walls of said each micro-orifice and themicro-region on the support together define a micro-well; positioning asecond layer to be in fluid-tight contact with an upper surface of thefirst layer, the second layer defining a pattern of macro-orifices, eachmacro-orifice of the pattern of macro-orifices having walls and defininga macro-region when the first layer is placed in fluid-tight contactwith the support and the second layer is placed in fluid-tight contactwith the first layer such that the walls of the macro-orifice and themacro-region together define a macro-well; each macro-regionencompassing at least one micro-region; immobilizing at least one cellof a plurality of cells in each respective micro-region on the supportso as to situate the at least one cell within a correspondingmicro-well, the cells thereby being arrayed on the support in a patternthat corresponds to the pattern of the micro-orifices; allowing thecells to grow to confluency within the micro-regions; providing at leastone of a plurality of test agents to at least one macro-well andallowing said test agent to contact confluent cells; removing said firstand second layer; monitoring cells for movement or lack of movement awayfrom said micro-regions; and correlating cellular movement or lack ofmovement away from said micro-regions with effect of said test agent oncellular movement.

[0056] Preferably each macro-region encompasses a plurality ofmicro-regions. A plurality of test agents can be provided into eachmacro-well.

[0057] The present invention also contemplates applying coating to anupper surface of the support before positioning said first layer. Thecoating is made of a material selected from the group consisting ofproteins, protein fragments, peptides, small molecules, lipid bilayers,metals, self-assembled monolayers, cells, extracellular matrix proteins,hydrogels, and matrigel.

[0058] In yet another embodiment, an assay comprises positioning a firstlayer to be in fluid-tight contact with a support, the first layerhaving an upper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well; immobilizing at least one cell of a plurality of cells ineach respective micro-region on the support so as to situate the atleast one cell within a corresponding micro-well, the cells therebybeing arrayed on the support in a pattern that corresponds to thepattern of the micro-orifices; allowing the cells to grow to confluencywithin the micro-regions; removing the first layer from the supportafter the step of immobilizing; positioning a second layer to be influid-tight contact with the support, the second layer defining apattern of macro-orifices, each macro-orifice of the pattern ofmacro-orifices having walls and defining a macro-region when the secondlayer is placed in fluid-tight contact with the support such that thewalls of the macro-orifice and the macro-region together define amacro-well; each macro-region encompassing at least one micro-region;providing at least one of a plurality of test agents to at least onemacro-well and allowing said test agent to contact confluent cells;removing said second layer; monitoring cells for movement or lack ofmovement away from said micro-regions; correlating cellular movement orlack of movement away from said micro-regions with effect of said testagent on cellular movement.

[0059] Another embodiment comprises the steps of: positioning a firstlayer to be in fluid-tight contact with a support, the first layerhaving an upper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well; positioning a second layer to be in fluid-tight contact withthe support, the second layer comprising a plurality of rings, the ringsdefining a pattern of respective macro-orifices, each ring having wallsand defining a macro-region when the second layer is placed influid-tight contact with the support such that the walls of the ring andthe macro-region together define a macro-well; each macro-regionencompassing at least one micro-region; immobilizing at least one cellof a plurality of cells in each respective micro-region on the supportso as to situate the at least one cell within a correspondingmicro-well, the cells thereby being arrayed on the support in a patternthat corresponds to the pattern of the micro-orifices, allowing thecells to grow to confluency within the micro-regions; providing at leastone of a plurality of test agents to at least one macro-well andallowing said test agent to contact confluent cells; removing said firstand second layer; monitoring cells for movement or lack of movement awayfrom said micro-regions; and correlating cellular movement or lack ofmovement away from said micro-regions with effect of said test agent oncellular movement.

[0060] In an alternate embodiment, an assay for monitoring cell movementcomprises the steps of: positioning a layer to be in fluid-tight contactwith the support, the layer defining a pattern of macro-orifices, eachmacro-orifice of the pattern of macro-orifices having walls and defininga macro-region when the layer is placed in fluid-tight contact with thesupport such that the walls of the macro-orifice and the macro-regiontogether define a macro-well; inserting each plug of a set of plugs in arespective macro-well, each of the plugs comprising a lower membraneplaced in fluid-tight contact with the support when the layer is placedin fluid-tight contact with the support and the plug is received in acorresponding macro-well defined by the layer and the support, the lowermembrane further defining a pattern of micro-orifices, wherein eachmicro-orifice has walls and defines a micro-region on the support whenthe plug is in fluid-tight contact with the support such that the wallsof the micro-orifice and the micro-region together define a micro-well;immobilizing a cell in at least one micro-region on the support so as tobe situated within the micro-well, such that the cell is arrayed on thesupport in a pattern that corresponds to the first pattern ofmicro-orifices; allowing the cells to grow to confluency within themicro-regions; providing at least one of a plurality of test agents toat least one macro-well and allowing said test agent to contactconfluent cells; removing said layer containing said plugs; monitoringcells for movement or lack of movement away from said micro-regions; andcorrelating cellular movement or lack of movement away from saidmicro-regions with effect of said test agent on cellular movement.

[0061] The present invention also provides for a system for monitoringcell movement comprising: a) a device for arraying cells comprising asupport; a first layer configured to be placed in fluid-tight contactwith the support, the first layer having an upper surface and defining apattern of micro-orifices, each micro-orifice of the pattern ofmicro-orifices having walls and defining a micro-region on the supportwhen the first layer is placed in fluid-tight contact with the supportsuch that the walls of said each micro-orifice and the micro-region onthe support together define a micro-well; a second layer configured tobe placed in fluid-tight contact with the upper surface of the firstlayer, the second layer defining a pattern of macro-orifices, eachmacro-orifice of the pattern of macro-orifices having walls and defininga macro-region when the first layer is placed in fluid-tight contactwith the support and the second layer is placed in fluid-tight contactwith the first layer such that the walls of the macro-orifice and themacro-region together define a macro-well; wherein the first layer andthe second layer are configured for an arraying of cells on the supportthrough the pattern of micro-orifices and the pattern of macro-orifices;allowing the cells to grow to confluency within the micro-regions;providing at least one of a plurality of test agents to at least onemacro-well and allowing said test agent to contact confluent cells;removing said first and second layer; monitoring cells for movement orlack of movement away from said micro-regions; and correlating cellularmovement or lack of movement away from said micro-regions with effect ofsaid test agent on cellular movement; b) an observation systemconfigured to observe movement or lack of movement of arrayed cells; andc) a controller configured to link coordinates movement of the devicefor arraying cells into said observation system.

[0062] The observation system preferably comprises a phase contrastmicroscope or a flourescent image microscope. The controller furthercomprises a computer interface configured to coordinate the movement ofthe device into the observation system. The observation system furthercomprises a recording device configured to record images of the cellsarrayed on the device for arraying cells. The recording devicepreferably comprises a digital camera configured to record images of thecells arrayed on the device for arraying cells, and wherein the recordedimages are in a digital output. The computer interface is preferablyconfigured to receive said digital output.

[0063] The present invention also provides for methods for monitoringand imaging cell growth. These methods involve positioning a first layerto be in fluid-tight contact with a support, the first layer having anupper surface and defining a pattern of micro-orifices, eachmicro-orifice of the pattern of micro-orifices having walls and defininga micro-region on the support when the first layer is placed influid-tight contact with the support such that the walls of said eachmicro-orifice and the micro-region on the support together define amicro-well. Further, a second layer is positioned to be in fluid-tightcontact with an upper surface of the first layer, the second layerdefining a pattern of macro-orifices, each macro-orifice of the patternof macro-orifices having walls and defining a macro-region when thefirst layer is placed in fluid-tight contact with the support and thesecond layer is placed in fluid-tight contact with the first layer suchthat the walls of the macro-orifice and the macro-region together definea macro-well; each macro-region encompassing at least one micro-region.

[0064] At least one cell of a plurality of cells is immobilized in eachrespective micro-region on the support so as to situate the at least onecell within a corresponding micro-well, the cells thereby being arrayedon the support in a pattern that corresponds to the pattern of themicro-orifices.

[0065] The cells are allowed to grow to confluency within themicro-regions. At least one of a plurality of test agents is provided toat least one macro-well. The test agent is allowed to contact confluentcells. Thereafter the first and second layer is removed. The cells arethen monitored for movement away from the micro-regions. The monitoringinvolves imaging the cells for at least two different time points togenerate an image for each of the at least two different time points togenerate at least two images, and calculating cellular movement from acomparison of the at least two images.

[0066] In yet other embodiments, cell growth and cell multiplication orproliferation is monitored and determined by a comparison of the atleast two images.

[0067] Another embodiment of the present invention provides an imageprocessing method comprising, from captured image data: a) creating afirst histogram of image data signal strength along a first axis of theimage data; b) identifying first coarse island locations from the firsthistogram; c) marking interstitial boundaries on the first axis betweenthe first coarse island locations; d) creating a second histogram ofimage data signal strength along a second axis of the image data; e)identifying second coarse island locations from the second histogram;and f) marking second interstitial boundaries on the second axis betweenthe second coarse island locations.

[0068] In another embodiment, the first and second coarse islandlocations are determined from maxima of the first and second histogramsrespectively. Alternatively, the first and second coarse islandlocations are determined from portions of the first and secondhistograms respectively that exceed a predetermined threshold value.

[0069] In another embodiment, the first and second interstitialboundaries are marked at midpoints between the first and second coarseisland locations respectively. Another embodiment involves defining aplurality of island bounding boxes based on the first and secondinterstitial boundaries.

[0070] Another embodiment of an imaging processing method of the presentinvention comprises, from source image data representing imaged cellularmaterial: for each pixel in a portion of the source image data;determining whether the source image data indicates the presence ofcellular material in a region of a scanning circle; and if so, settingimage data for a co-located, similarly dimensioned scanning circle insecond image data; and thereafter, identifying objects based on thesecond image data. Further, a bounding box for each object identified inthe image data may be defined.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The present invention is illustrated by way of example and notlimitation in the figures in the accompanying drawings, in which likereferences indicate similar elements.

[0072]FIG. 1(a) is a perspective view of a qualitative cell migrationsystem, in accordance with an example embodiment of the presentinvention.

[0073]FIG. 1(b) is a cross-sectional view of the qualitative cellmigration assay plate shown in FIG. 1(a), taken along the lines II-II.

[0074]FIG. 2(a) is a perspective view of a qualitative cell migrationsystem, in accordance with an example embodiment of the presentinvention.

[0075]FIG. 2(b) is a cross-sectional view of the qualitative cellmigration assay plate shown in FIG. 2(a), taken along the lines IV-IV.

[0076]FIG. 3(a) is a top view of a support for a qualitative cellmigration system, in accordance with one embodiment of the presentinvention.

[0077]FIG. 3(b) is a side view of the support shown in FIG. 3(a).

[0078]FIG. 4(a) is a top view of a first layer for a qualitative cellmigration system, in accordance with one embodiment of the presentinvention.

[0079]FIG. 4(b) is a side view of the first layer shown in FIG. 4(a).

[0080]FIG. 5(a) is a top view of a second layer for a qualitative cellmigration system, in accordance with one embodiment of the presentinvention.

[0081]FIG. 5(b) is a side view of the second layer shown in FIG. 5(a).

[0082]FIG. 6(a) is a top view of a first layer for a qualitative cellmigration system, in accordance with one embodiment of the presentinvention.

[0083]FIG. 6(b) is a top view of a second layer for a qualitative cellmigration system, in accordance with one embodiment of the presentinvention.

[0084]FIG. 6(c) is a top view of the second layer shown in FIG. 6(b)positioned on the first layer shown in FIG. 6(a).

[0085]FIG. 7(a) is a top view of a first layer for a qualitative cellmigration system, in accordance with another embodiment of the presentinvention.

[0086]FIG. 7(b) is a top view of a second layer for a qualitative cellmigration system, in accordance with another embodiment of the presentinvention.

[0087]FIG. 7(c) is a top view of the second layer shown in FIG. 7(b)positioned on the first layer shown in FIG. 7(a).

[0088]FIG. 8(a) is a top view of a qualitative cell migration assayplate, in accordance with another embodiment of the present invention.

[0089]FIG. 8(b) is a cross-sectional view of the qualitative cellmigration assay plate shown in FIG. 8(a), taken along the lines IX-IX.

[0090]FIG. 8(c) is a top view of a qualitative cell migration assayplate, in accordance with another embodiment of the present invention.

[0091]FIG. 8(d) is a cross-sectional view of a plug insertable into thequalitative cell migration assay plate shown in FIG. 8(c).

[0092]FIG. 9(a) is a perspective view of a PDMS casting having aplurality of macroposts disposed thereon, in accordance with oneembodiment of the present invention.

[0093]FIG. 9(b) is a perspective view of a 96-well microtiter plate thatmay be employed for casting the macrocosms shown in FIG. 9(a).

[0094] FIGS. 10(a) through 10(c) illustrates steps that may be performedin order to fabricate first and second elastomeric layers, in accordancewith one embodiment of the present invention.

[0095] FIGS. 11(a) through 11 (c) illustrates steps that may beperformed in order to fabricate first and second elastomeric layers, inaccordance with another embodiment of the present invention.

[0096]FIG. 12(a) illustrates a first cell type patterned intomicro-orifices, in accordance with one embodiment of the presentinvention.

[0097]FIG. 12(b) illustrates a second type of cells arrayed around thefirst cell type shown in FIG. 12(a).

[0098]FIG. 12(c) illustrates an overlayed arrangement of the first andsecond cell types shown in FIGS. 12(a) and 12(b).

[0099]FIG. 13(a) illustrates a second layer positioned on a first layer,in accordance with another embodiment of the present invention.

[0100]FIG. 13(b) illustrates the first and second layers shown in FIG.13(a) having cells patterned therethrough onto a support.

[0101]FIG. 13(c) illustrates the first layer shown in FIG. 13(b) beingremoved such that the cells arrayed on the support shown in FIG. 13(b)are permitted to migrate.

[0102]FIG. 13(d) illustrates cells that have been patterned through thefirst and second layers shown in FIG. 13(b) onto a support and that havegrown to confluence.

[0103]FIG. 13(e) shows the cells having migrated upon the removal of thefirst and second layers, as shown in FIG. 13(c).

[0104]FIG. 14(a) illustrates a self-assembled monolayer having a“switchable head,” in accordance with one embodiment of the presentinvention.

[0105]FIG. 15(a) illustrates the effect of a test agent on cell motilityfor a control group and a particular cell type, in accordance with oneembodiment of the present invention.

[0106]FIG. 15(b) is a graphical representation of the effects of thetest agent on cell motility as shown in FIG. 15(a).

[0107]FIGS. 16 and 17 illustrate the effect of a various agents on cellmotility for a group of cell, in accordance with various embodiments ofthe present invention.

[0108]FIG. 18 is a graphical representation of the amount of cellmotility relative to an amount of cell proliferation, in accordance withone embodiment of the present invention.

[0109]FIG. 19 is a schematic diagram of a system for measuring themigration or motility of cells, in accordance with one embodiment of thepresent invention.

[0110]FIG. 20 contains the pictorial results of an assay using aqualitative cell motility assay plate showing farnesyl transferaseinhibition in MS 1 and SVR cells, in accordance with one embodiment ofthe present invention.

[0111]FIG. 21 shows the data analysis of cell motility of MS 1 and SVRaffected by farnesyl transferase inhibition, in accordance with oneembodiment of the present invention.

[0112]FIG. 22 shows graphs of the results of an assay determining theinhibition of 769-P motility using MMP inhibitor GM6001, and a chartcomparing the results of the assay performed in a transwell system andthe assay performed in employing the qualitative cell motility assayplate, in accordance with one embodiment of the present invention.

[0113]FIG. 23 presents the results of an assay where the effects ofseveral inhibitors in the RAS pathway were measured, in accordance withone embodiment of the present invention.

[0114]FIG. 24 depicts a cell motility assay wherein cells are patternedin a predetermined area using a physical constraint. The physicalconstraint is removed and cell motility is monitored. Well definedpatterns of cells can be created once the membrane is lifted.

[0115]FIG. 25 depicts a particular cell motility assay using endothelialcells and agonists and antagonists of such cells. The results depictedshow that cell motility is affected when an inhibitor to VEGF is added.Normally VEGF stimulates cells to migrate as depicted in FIG. 25A. InFIG. 25B, the concentration of VEGF is fixed. An antibody to VEGF isadded in various concentrations. The motility of cells is affected in adose-dependent fashion by the antibody. When an inhibitor to the VEGFreceptor is added, the cells migrate at a such slower rate as depictedin FIG. 25C. FIG. 25D shows that cell motility can be affected withkinase inhibitors. Normally enzyme inhibition screens involve proteinsand their substrates and not cells. FIG. 25D depicts enzyme inhibitionscreens in a cell based context.

[0116]FIG. 26 shows that cell migration may be affected by the supportupon which the cells are placed. It also depicts the use of fibronectinon the support as a cytophilic substance to encourage adherence of cellsto the support.

[0117]FIG. 27 depicts control of cell cycle by patterning.

[0118]FIG. 28 depicts the effects of cell patterning geometry on celldifferentiation.

[0119]FIG. 29 depicts cell differentiation brought about by patterningof the cells into certain constraints.

[0120]FIG. 30 depicts single cell patterning and the subsequentevaluation of cytoskeletal stability and rearrangement.

[0121]FIG. 31 illustrates a flow chart of a assay according to anembodiment of the present invention.

[0122]FIG. 32 illustrates exemplary test apparatus according to anembodiment of the present invention.

[0123]FIG. 33 illustrates exemplary test apparatus according to anotherembodiment of the present invention.

[0124]FIG. 34 illustrates a method of performing island acquisitionaccording to an embodiment of the present invention.

[0125]FIG. 35 illustrates idealized, exemplary image data for use in anembodiment of the present invention.

[0126]FIG. 36 illustrates idealized, exemplary image data for use in anembodiment of the present invention.

[0127]FIG. 37 illustrates idealized, exemplary image data for use in anembodiment of the present invention.

[0128]FIG. 38 illustrates a method of identifying islands according toan embodiment of the present invention.

[0129]FIG. 39 is a screen shot of exemplary source image data.

[0130]FIG. 40 is a screen shot of exemplary dilated image data.

[0131]FIG. 41 depicts various images of data and digital imagesretrieved from an assay according to an embodiment of the presentinvention. Algorithms are used to convert digital images into computerreadable data that is then converted into usable graphic interfaces.

DETAILED DESCRIPTION

[0132]FIG. 1(a) is a schematic, perspective view of a qualitative cellmigration system 190 in accordance with an embodiment of the presentinvention. The qualitative cell migration system 190 includes aqualitative cell migration assay plate 100, an observation system 110,and a controller 120. The controller 120 in this embodiment is in signalcommunication with the observation system 110 via line 130. Thecontroller 120 and the observation system 110 may be positioned andprogrammed to observe, record, and analyze the migration, movement, andbehavior of cells that are placed in or on the qualitative cellmigration assay plate 100, as readily recognizable by a person skilledin the art.

[0133] The present invention provides a cell migration assay plate 100for the quantification of the qualitative cell patterning and migration.Embodiments of the assay plate, according to the present invention,allow a patterning of cells in a discrete, predetermined array. Thepresent invention also provides cell migration/motility assays, alsoreferred to as “CMAs,” which preferably uses a qualitative cellmigration assay plate according to the present invention to patterncells into discrete arrays and uses a cell migration system according tothe present invention to monitor and record the results of the assays.Embodiments of the cell migration assay plate, the cell migrationsystem, and the cell migration/motility assays of the present inventionare compatible with the demands of high-throughput screening, andrepresent a significant advance in both throughput and ease of use.Generally, with embodiments of the qualitative cell migration assayplate of the invention, cells are patterned into a specific geometry,treated with various cell affecting agents, and allowed to migrate orotherwise react in response to a cell affecting agent.

[0134]FIG. 1(b) is a cross-sectional view of cell migration assay plate100 of FIG. 1(a), taken along lines II-II. Embodiments of the cellmigration assay plate according to the present invention, as shown byway of example in the embodiments of FIGS. 1(a) and 1(b), include: asupport 140 onto which cells may be arrayed, a first layer 150 thatprovides a pattern through which cells may be arrayed on the support140; and a second layer 160. The support 140 provides a base upon whichcells can be patterned, attached, or reversibly or irreversiblyimmobilized. The support 140 has an upper surface 140 a. The first layer150 defines a plurality of orifices 300 therethrough, referred tohereinafter as “micro-orifices 300.” The micro-orifices 300 are arrangedin a pattern or array that defines positions in which cells may bedeposited, attached, or reversibly or irreversibly immobilized to theupper surface 140 a of the support 140. The micro-orifices 300 havewalls 150 a that define the micro-orifices 300. The second layer 160defines a plurality of orifices 170 therethrough, referred tohereinafter as “macro-orifices 170.” The macro-orifices 170 are arrangedin a pattern or array through which test agents or solutions aredeposited to contact cells that were previously deposited, attached, orreversibly or irreversibly immobilized to the upper surface 140 a of thesupport 140. The macro-orifices 170 have walls 160 a that define themacro-orifices 170.

[0135] The size of the support 140 preferably matches the dimensions ofan industry standard micro-titer plate. For example, FIGS. 3(a) and 3(b)illustrate the support 140, according to one embodiment of the presentinvention. More specifically, FIG. 3(a) is a plan view that illustratesthe support 140 having a length dimension L and a width dimension W.According to one embodiment, the length dimension L of the support 140is approximately 3 inches (75 mm), while the width dimension W isapproximately 5 inches (125 mm). Preferably, all of the layers of thecell migration assay plate 100 would have corresponding outer dimensionsand would be amenable to use in standard laboratory platforms such asmicrotiter plate readers, automatic handlers, and fluid deliverysystems.

[0136] Referring to the embodiment illustrated in FIG. 1(b), themicro-orifices 300 extend through the entire thickness of the firstlayer 150. In a preferred embodiment of the present invention, the firstlayer 150 defines an array of micro-orifices 300, which are disposed inan array of discrete first positions. In addition, the first layer 150is preferably capable of making conformal contact, that is, aform-fitting fluid-tight contact, with support 140, when brought intocontact with the support 140. Furthermore, the first layer 150 ispreferably capable of self-sealing to the support 140, e.g., creating aseal with the support 140 without the use of a sealing agent. When thefirst layer 150 is brought into contact with the support 140 to create afluid-tight seal, a plurality of wells, referred to hereinafter as“micro-wells,” are formed. The walls of each micro-well 141 are definedby the walls 150 a of the micro-orifices 300 in the first layer 150,while the bottom of each micro-well 141 is defined by an exposed regionon the upper surface 140 a of the support 140. Advantageously, eachmicro-well 141 is individually fluidically addressable, e.g., may have adifferent fluid introduced therein.

[0137] The first layer 150 may be comprised of materials commonly usedin biological sciences, such as glass, elastomers (e.g., PDMS), rigidplastics (e.g., polyethylene, polypropylene, polystyrene, polycarbonate,PMMA), metals, silicon, silicon dioxide and other rigid supports.

[0138] According to one embodiment of the present invention, the firstlayer 150 may be treated, conditioned or coated with a substance thatresists cell attachment so that when the first layer 150 is lifted fromthe support, the risk of damaging cells is reduced. Coatings resistantto proteins are known in the art and include, but are not limited to:bovine serum albumin (BSA), gelatin, lysozyme, octoxynol, polysorbate 20(polyoxyethylenesorbitan monolaurate), and polyethylene oxide-containingblock copolymer surfactants. Conversely, according to other embodimentsof the present invention, the first layer 150 is not so coated, suchthat when the first layer 150 is removed, the cells that have adhered tothe first layer 150 will likely be damaged as the first layer 150 ispeeled away from the support. By damaging cells, phenomena, such aswound healing, may be observed. 11391 FIG. 4(a) illustrates the firstlayer 150 defining a plurality of micro-orifices 300 disposedtherethrough. In the embodiment shown, the micro-orifices 300 aregrouped into discrete areas. These discrete areas may have a variety ofshapes and sizes. In the embodiment shown, each area has a cluster ofmicro-orifices 300 arranged in a circular arrangement. It is understoodthat the micro-orifices 300 of the first layer 150 may have any otherarrangement that would be within the knowledge of a person skilled inthe art, such as, for example, a rectangular, hexagonal, circular or anyanother arrangement.

[0139] The diameter of the micro-orifices 300 (and also the diameter ofthe micro-wells 141 that are defined by the walls 150 a of themicro-orifices), shown as dimension “d” in FIG. 4(a), may be variedaccording to cell types and the desired number of cells to be placedinto each micro-well 141. For example, if the diameter of the micro-well141 and the cell to be placed in the micro-well 141 are both 10 mm, onlyone cell will be depositable through each micro-orifice 300 and intoeach micro-well 141. Thus, in this example, if the diameter of themicro-orifice 300 is 100 mm, up to approximately 100 cells may bedeposited in a micro-well 141 defined by that micro-orifice 300.

[0140] According to embodiments of the present invention, the diameter dof micro-wells 141 varies from about 1 mm to about 500 mm, and ispreferably from about 40 mm to about 200 mm. In most cases, the diameterd is greater than the diameter of cells used in experiments, but inspecialized assays, the diameter d may be smaller than that of thecells. For example, if it is desired to pattern a single cell througheach micro-orifice 300 of the first layer 150 and into micro-well 141,the diameter d may range from about 1 microns to about 20 microns. In atypical chemotaxis assay, the diameter d is preferably approximately0.3-0.8 times the diameter of cells. Furthermore, the distance betweenadjacent micro-orifices 300 (and thus the distance between adjacentmicro-wells 141 defined by the micro-orifices 300) may be varied. Thisdistance is identified as dimension “p” in FIG. 4(a). Although anydistance p may be employed, this distance p may vary, according tovarious example embodiments of the present invention, from about thesame distance as the diameter dimension d to about 10 times the diameterd.

[0141] The second layer 160 is comprised of materials commonly used inbiological sciences, such as glass, elastomers (e.g., PDMS), rigidplastics (e.g., polyethylene, polypropylene, polystyrene, polycarbonate,PMMA), metals, silicon, silicon dioxide and other rigid supports. Apreferred material is PDMS, and a more preferred material is acombination of PDMS and a rigid plastic such as polycarbonate.

[0142] Referring to the embodiment illustrated in FIG. 1(b), themacro-orifices 170 extend through the entire thickness of the secondlayer 160. In a preferred embodiment of the present invention, thesecond layer 160 has an array of macro-orifices 170. In addition, thesecond layer 160 is preferably capable of making conformal contact, thatis, a form-fitting, fluid tight contact when brought into contact witheither an upper surface 150 b of the first layer 150, or the uppersurface 140 a of the support 140. Furthermore, the second layer 160 ispreferably capable of self-sealing to either of upper surface 150 b orupper surface 140 a, e.g., creating a conformal, fluid-tight sealtherewith without the use of a sealing agent. In the embodiment of thepresent invention shown in FIG. 1(b), when the second layer 160 isbrought into contact with the upper surface 150 b of the first layer 150to create a fluid-tight seal, a plurality of wells 151, referred tohereinafter as “macro-wells 151,” are formed. The walls of eachmacro-well 151 are defined by the walls 160 a of the macro-orifices 170in the second layer 160. The bottom of each macro-well 151 is theexposed region defined by the size and shape of the macro-orifice 151 atthe lower surface 161 of the second layer 160. For instance, in theembodiment illustrated in FIG. 1(b), the bottom of the macro-well 151 isthe exposed region defined by a portion of the upper surface 140 a ofthe support 140, the walls 150 a of the micro-orifices 300 that areencompassed by the macro-well 151, and by the exposed regions of theupper surface 150 b of the first layer 150 within the encompassedmicro-wells 300. Thus, as should be evident, the elements that make upthe bottom of the macro-wells 151 depend on the size and orientation ofthe macro-wells 151 relative to the micro-wells 141. Advantageously,each micro-well 141 is individually fluidically addressable, e.g., mayhave a different fluid introduced therein. It is also noted that, inaccordance with an alternate embodiment of the present invention, thefirst layer 150 is removed from the support 140 after arraying the cellsthrough the micro-orifices 300, and the second layer 160 is brought intocontact with the upper surface 140 a of the support 140. In this case,the bottom of the macro-well 151 is an exposed region of the uppersurface 140 a of the support 140, and may encompass cells or groups ofcells that were previously arrayed onto the upper surface 140 a of thesupport 140.

[0143] The macro-wells 151 defined by the macro-orifices 170 mayencompass discrete regions of the first layer 150 such that fluids addedto one macro-orifice 170 will flow to the encompassed micro-wells 141,but may not flow to adjacent or other micro-wells 141 not encompassed bythe macro-well 151. In this embodiment, the macro-wells 151 allow foreasy addition and removal of solutions, while the first layer 150 ofmicro-orifices 300 provides the spatial patterning of the cells.

[0144] As previously mentioned, the micro-orifices 300 may be sized toaccommodate the passage of several cells at a time, the passage of asingle cell at a time, or the passage of a portion of a cell. The sizeof the micro-orifices 300 may be selected to accommodate the particularcell and stimulus being studied. Depending on the size and orientationof the micro-orifices 300, cells can be placed in specific regions,groups or patterns on the support layer 140. In so doing, the startingpoint of each cell or cell group can be readily identified and itsdistance of travel readily measured and timed for various time periods.Preferably, more than one cell will settle through each orifice.

[0145]FIG. 5 illustrates the second layer 160 having a plurality ofmacro-orifices 170 defined therethrough. In the embodiment shown, themacro-orifices 170 are circular in a top plan view thereof, although itis understood that the macro-orifices 170 may have a variety of shapesand sizes. The number of macro-wells 151, the diameter of themacro-orifices 170 (and also the diameter of the macro-wells 151 thatare defined by the walls 160 a of the micro-orifices 170), shown asdimension “d” in FIG. 5, and the distance between adjacent macro-wells151, shown as dimension “p” in FIG. 5, may each be varied according tocell types and the number of micro-wells 141 desired to be encompassedin each macro-well 151, or the process desired to be performed.Preferably the arrangement of the macro-orifices 300, and thus thearrangement of the macro-wells 151 defined thereby, corresponds to thefootprint of standard 24-, 96-, 384-, and 1536-well micro-titer plates.For example, the typical dimensions of various standard micro-titerplates (“ID” refers to the inner diameter of a well of the micro-titerplate, while “p” refers to the distance between adjacent wells) are asfollows: Device ID (mm) p (mm)  24 well 9-15 18  96 well 6 9  384 well 34.5 1536 well 1.5 2.25

[0146] In one embodiment of the present invention, the second layer 160is comprised of an elastomer, such as PDMS. In this embodiment, themacro-orifices 170 are formed in the second layer 160 in a manner thatis similar to the manner in which the micro-orifices 300 are formed inthe first layer 150, e.g., precursor PDMS is spin cast on to a masterhaving posts corresponding in size (diameter and length) and pitch asthe desired macro-orifices. In another embodiment, the second layer 160is comprised of a rigid material including, but not limited to, glass,rigid plastics or metals. The macro-orifices 170 are formed in thesematerials by methods known in the art, such as molding, etching, andpunching.

[0147] In various other embodiments of the present invention,macro-orifices 170 of the second layer 160 may comprise individual ringsor interconnected rings. For instance, in one embodiment, the secondlayer 160 comprises rings made of a rigid plastic such as polypropylene,and having a diameter equal to the desired diameter of themacro-orifices 170. The rigid rings may be molded together with anelastomer such as PDMS to form the second layer 160.

[0148] The thickness or height of the first layer 150, which is shown inFIG. 4(b) and which is designated as “2h,” may be predetermined so as toaccommodate a desired number of cells, e.g., a single cell or multiplecells. In other words, the thickness of the first layer 150 dictates themaximum depth of the micro-wells 141 formed by the micro-orifices 300.To alter the thickness of the first layer 150, one may stack identicalfirst layer 150 s on top of each other to achieve the desired thickness.In alternate embodiments, the first layer 150 may be fabricated so as tohave a desired thickness. Because elastomers such as PDMS create aconformal contact that is reversible, stacking of the layers allows oneto achieve a micro-orifice of a desired depth. By “reversible,” what ismeant in the context of the present invention, is a conformal contactthat can be undone without compromising a structural integrity of thecomponent making the conformal contact.

[0149] The thickness or height of the support 140, shown in FIG. 3(b)and designated as “h,” may be chosen as desired. Similarly, thethickness or height of the second layer 160, as shown in FIG. 5(b) anddesignated as “h,” may be chosen to accommodate a desired amount ofsolution to be added into the macro-wells 151 formed by themacro-orifices 170. A preferred height “h” of the second layer 160ranges from about 2 mm to about 12 mm.

[0150] The support 140 on which the cells may be placed or patternedcomprises a material that is compatible with the cells. Suitablematerials may include standard materials used in cell biology, such asglass, ceramics, metals, polystyrene, polycarbonate, polypropylene, aswell as other plastics including polymeric thin films, and polymethylmethacrylate (PMMA). Preferably, the material provides sufficientrigidity to allow the device to be handled either manually or byautomatic laboratory handlers. A preferred material is optical gradepolycarbonate with a thickness of about 0.2 to 2 mm, as this may allowthe viewing of the patterned cells with optical microscopy techniques.

[0151] Additionally, the support 140 may be comprised of any materialthat provides a conformal contact with additional layers of the cellmigration assay plate 100. Materials which allow conformal contact areknown in the art and include elastomers with a preferred elastomer beingpolydimethylsiloxane (“PDMS”). In an alternate embodiment, the support140 and/or the first layer 150 are comprised of an elastomer. Elastomerssuch as PDMS are preferred in that the conformal contact prevents fluidsfrom infiltrating other orifices in the first layer 150 or the secondlayer 160. In other embodiments, sealing agents or mechanical sealingdevices such as clamps and gaskets may also be used to create or enhancethe seal between the support 140 and the first layer 150 or between thefirst layer 150 and the second layer 160. Sealing agents capable ofcreating fluid-tight seals between two materials are known in the artand include glues, inert gels, and swellable resins.

[0152]FIG. 2(a) illustrates one embodiment wherein support 210 istreated with a coating 220. A cross-sectional view of the embodimentshown in FIG. 2(a), taken along the lines II-II, is shown in FIG. 2(b).Alternatively, support 210 may be overlayed with a membrane having adesired treatment or coating 220 thereon.

[0153] Coating 220 may be made of any substance that achieves a desiredeffect on the cells to be arrayed or may be made of any substance toassist in the arraying of the cells or it may comprise a bio-inertcoating. Coating 220 may also comprise proteins, proteins fragments,peptides, small molecules, lipid bilayers, metals, or self-assembledmonolayers. Self-assembled monolayers are the most widely studied andbest developed examples of nonbiological, self-assembling systems. Theyform spontaneously by chemisorption and self-organization offunctionalized, long-chain organic molecules onto the surfaces ofappropriate substrates. Self-assembled monolayers are usually preparedby immersing a substrate in the solution containing a ligand that isreactive toward the surface, or by exposing the substrate to the vaporof the reactive species. There are many systems known in the art toproduce self-assembled monolayers.

[0154] The best characterized systems of self-assembled monolayers arealkanethiolates CH₃(CH₂)_(n)S— on gold. Alkanethiols chemisorbspontaneously on a gold surface from solution and form adsorbedalkanethiolates. Sulfur atoms bonded to the gold surface bring the alkylchains in close contact—these contacts freeze out configurationalentropy and lead to an ordered structure. For carbon chains of up toapproximately 20 atoms, the degree of interaction in a self-assembledmonolayer increases with the density of molecules on the surface and thelength of the alkyl backbones. Only alkanethiolates with n>11 form theclosely packed and essentially two-dimensional organic quasi-crystalssupported on gold that characterize the self-assembled monolayers mostuseful in soft lithography. The formation of ordered self-assembledmonolayers on gold from alkanethiols is a relatively fast process.Highly ordered self-assembled monolayers of hexanedecanethiolate on goldcan be prepared by immersing a gold substrate in a solution ofhexadecanethiold in ethanol (ca. 2 mM) for several minutes, andformation of self-assembled monolayers during microcontact printing mayoccur in seconds.

[0155] It may be desirable to pattern the self-assembled monolayer tohave an arrayed surface. For example, it may be desirable to pattern theself-assembled monolayer such that it has an array matching the array ofmicro-orifices or macro-orifices or any other array. Patterningself-assembled monolayers in the plane of the surface has been achievedby a wide variety of techniques, including micro-contact printing,photo-oxidation, photo-cross-linking, photo-activation,photolithography/plating, electron beam writing, focused ion beamwriting, neutral metastable atom writing, SPM lithography,micro-machining, micro-pen writing. A preferred method is micro-contactprinting. Micro-contact printing is described, by way of example, inU.S. Pat. No. 5,776,748, which is herein incorporated by reference inits entirety.

[0156] In another embodiment, coating 220 comprising self-assembledmonolayers is “patterned” by micro-contact printing. The self-assembledmonolayer patterns are applied to the support using a stamp in a“printing” process in which the “ink” consists of a solution including acompound capable of chemisorbing to form a self-assembled monolayer. Theink is applied to the surface of a plate using the stamp and deposits aself-assembled monolayer on the support in a pattern determined by thepattern on the stamp. The support may be stamped repeatedly with thesame or different stamps in various orientations and with the same ordifferent self-assembled monolayer-forming solutions. In addition, afterstamping, the portions of the support which remain bare or uncovered bya self-assembled monolayer may be derivatized. Such derivatization mayconveniently include exposure to another solution including aself-assembled monolayer-forming compound. The self-assembledmonolayer-forming or derivatizing solutions are chosen such that theregions of the finished support defined by the patterns differ from eachother in their ability to bind biological materials. Thus, for example,a grid pattern may be created in which the square regions of the gridare cytophilic and bind cells but the linear regions of the grid arecytophobic and no cells bind to these regions.

[0157] A simple illustration of the general process of microcontactprinting is provided by way of example below. A polymeric material iscast onto a mold with raised features defining a pattern to form astamp. The stamp with the stamping surface after curing is separatedfrom the mold. The stamp is inked with a desired “ink,” which includes aself-assembled monolayer-forming compound. The “inked” stamp is broughtinto contact with a plate comprising a substrate and optionally, coatedwith a thin coating of surface material. The self-assembled monolayerforming compound of the ink chemisorbs to the material surface to form aself-assembled monolayer with surface regions in a pattern correspondingto the stamping surface of the stamp. The plate can then be exposed to asecond or filling solution including a self-assembled monolayer-formingcompound. The second solution has filled the bare regions of the surfacematerial with a second or filling self-assembled monolayer. The platehaving the patterned self-assembled monolayer can then have a materialselectively bound to the surface regions of the first self-assembledmonolayer but not bound the surface regions of the second self-assembledmonolayer and vice-versa.

[0158] The stamp is inked with a solution capable of forming aself-assembled monolayer by chemisorption to a surface. The inking may,for example, be accomplished by: (1) contacting the stamp with a pieceof lint-free paper moistened with the ink; (2) pouring the ink directlyonto the stamp or; (3) applying the ink to the stamp with a cotton swab.The ink is then allowed to dry on the stamp or is blown dry so that noink in liquid form, which may cause blurring, remains on the stamp. Theself-assembled monolayer-forming compound may be very rapidlytransferred to the stamping surface. For example, contacting thestamping surface with the compound for a period of time of approximatelytwo seconds is generally adequate to effect sufficient transfer, orcontact may be maintained for substantially longer periods of time. Theself-assembled monolayer-forming compound may be dissolved in a solventfor such transfer, and this is often advantageous in the presentinvention. Any organic solvent within which the compound dissolves maybe employed but, preferably, one is chosen which aids in the absorptionof the self-assembled monolayer-forming compound by the stampingsurface. Thus, for example, ethanol, THF, acetone, diethyl ether,toluene, isooctane and the like may be employed. For use with a PDMSstamp, ethanol is particularly preferred, and toluene and isooctane andnot preferred as they are not well absorbed. The concentration of theself-assembled monolayer-forming compound in the ink solution may be aslow as 1 μM. A concentration of 1-10 mM is preferred and concentrationsabove 100 mM are not recommended.

[0159] The support is then contacted with the stamp such that the inkedstamping surface bearing the pattern contacts the surface material ofthe plate. This may be accomplished by hand with the application ofslight finger pressure or by a mechanical device. The stamp and plateneed not be held in contact for an extended period; contact timesbetween 1 second and 1 hour result in apparently identical patterns forhexadecanethiol (1-10 mM in ethanol) ink applied to a plate with a goldsurface. During contact, the self-assembled monolayer-forming compoundof the ink reacts with the surface of the plate such that, when thestamp is gently removed, a self-assembled monolayer is chemisorbed tothe plate in a pattern corresponding to the stamp.

[0160] A variety of compounds may be used in solution as the ink and avariety of materials may provide the surface material onto which the inkis stamped and the self-assembled monolayer is formed. In general, thechoice of ink will depend on the surface material to be stamped. Ingeneral, the surface material and self-assembled monolayer-formingcompound are selected such that the self-assembled monolayer-formingcompound terminates at a first end in a functional group that binds orchemisorbs to the surface of the surface material. As used herein, theterminology “end” of a compound is meant to include both the physicalterminus of a molecule as well as any portion of a molecule availablefor forming a bond with the surface in a way that the compound can forma self-assembled monolayer. The compound may comprise a molecule havingfirst and second terminal ends, separated by a spacer portion, the firstterminal end comprising a first functional group selected to bond to thesurface material of the plate, and the second terminal end optionallyincluding a second functional group selected to provide a self-assembledmonolayer on the material surface having a desirable exposedfunctionality. The spacer portion of the molecule may be selected toprovide a particular thickness of the resultant self-assembledmonolayer, as well as to facilitate self-assembled monolayer formation.Although self-assembled monolayers of the present invention may vary inthickness, as described below, self-assembled monolayers having athickness of less than about 50 Angstroms are generally preferred, morepreferably those having a thickness of less than about 30 Angstroms andmore preferably those having a thickness of less than about 15Angstroms. These dimensions are generally a function of the selection ofthe compound and in particular the spacer portion thereof.

[0161] A wide variety of surface materials and self-assembledmonolayer-forming compounds are suitable for use in the presentinvention. A non-limiting exemplary list of combinations of surfacematerials and functional groups which will bind to those surfacematerials follows. Although the following list categorizes certainpreferred materials with certain preferred functional groups whichfirmly bind thereto, many of the following functional groups would besuitable for use with exemplary materials with which they are notcategorized, and any and all such combinations are within the scope ofthe present invention. Preferred materials for use as the surfacematerial include metals such as gold, silver, copper, cadmium, zinc,palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten,and any alloys of the above when employed with sulfur-containingfunctional groups such as thiols, sulfides, disulfides, and the like;doped or undoped silicon employed with silanes and chlorosilanes; metaloxides such as silica, alumina, quartz, glass, and the like employedwith carboxylic acids; platinum and palladium employed with nitrites andisonitriles; and copper employed with hydroxamic acids. Additionalsuitable functional groups include acid chlorides, anhydrides, sulfonylgroups, phosphoryl groups, hydroxyl groups and amino acid groups.Additional surface materials include germanium, gallium, arsenic, andgallium arsenide. Additionally, epoxy compounds, polysulfone compounds,plastics and other polymers may find use as the surface material in thepresent invention. Polymers used to form bioerodable articles, includingbut not limited to polyanhydrides, and polylactic and polyglycolicacids, are also suitable. Additional materials and functional groupssuitable for use in the present invention can be found in U.S. Pat. No.5,079,600, issued Jan. 7, 1992, which is incorporated herein in itsentirety by reference.

[0162] According to a particularly preferred embodiment of the presentinvention, a combination of gold as the surface material and aself-assembled monolayer-forming compound having at least onesulfur-containing functional group such as a thiol, sulfide, ordisulfide is selected.

[0163] The self-assembled monolayer-forming compound may terminate in asecond end or “head group,” opposite to the end bearing the functionalgroup selected to bind to the surface material, with any of a variety offunctionalities. That is, the compound may include a functionality that,when the compound forms a self-assembled monolayer on the surfacematerial, is exposed. Such a functionality may be selected to create aself-assembled monolayer that is hydrophobic, hydrophilic, thatselectively binds various biological or other chemical species, or thelike. For example, ionic, nonionic, polar, nonpolar, halogenated, alkyl,aryl or other functionalities may exist at the exposed portion of thecompound. A non-limiting, exemplary list of such functional groupsincludes those described above with respect to the functional group forattachment to the surface material in addition to: —OH, —CONH—,—CONHCO—, —NH₂, —NH—, —COOH, —COOR, —CSNH—, —NO₂ ⁻, —SO₂ ⁻, —RCOR—,—RCSR—, —RSR, —ROR—, —PO₄ ⁻³, —OSO₃ ⁻², —SO₃ ⁻, —NH_(x)R₄-x⁺, —COO⁻,—SOO⁻, —RSOR—, —CONR₂, —(OCH₂ CH₂)_(n) OH (where n=1-20, preferably1-8), —CH₃, —PO₃H⁻, —2-imidazole,—N(CH₃)₂, —NR₂, —PO₃H₂, —CN,—(CF₂)_(n)CF₃ (where n=1-20, preferably 1-8), olefins, and the like. Inthe above list, R is hydrogen or an organic group such as a hydrocarbonor fluorinated hydrocarbon. As used herein, the term “hydrocarbon”includes alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl,and the like. The hydrocarbon group may, for example, comprise methyl,propenyl, ethynyl, cyclohexyl, phenyl, tolyl, and benzyl groups. Theterm “fluorinated hydrocarbon” is meant to refer to fluorinatedderivatives of the above-described hydrocarbon groups.

[0164] In addition, the functional group may be chosen from a widevariety of compounds or fragments thereof which will render theself-assembled monolayer generally or specifically “biophilic” as thoseterms are defined below. Generally biophilic functional groups are thosethat would generally promote the binding, adherence, or adsorption ofbiological materials such as, for example, intact cells, fractionatedcells, cellular organelles, proteins, lipids, polysaccharides, simplecarbohydrates, complex carbohydrates, and/or nucleic acids. Generallybiophilic functional groups include hydrophobic groups or alkyl groupswith charged moieties such as —COO⁻, —PO₃H⁻ or 2-imidazolo groups, andcompounds or fragments of compounds such as extracellular matrixproteins, fibronectin, collagen, laminin, serum albumin, polygalactose,sialic acid, and various lectin binding sugars. Specifically biophilicfunctional groups are those that selectively or preferentially bind,adhere or adsorb a specific type or types of biological material so as,for example, to identify or isolate the specific material from a mixtureof materials. Specific biophilic materials include antibodies orfragments of antibodies and their antigens, cell surface receptors andtheir ligands, nucleic acid sequences and many others that are known tothose of ordinary skill in the art. The choice of an appropriatebiophilic functional group depends on considerations of the biologicalmaterial sought to be bound, the affinity of the binding required,availability, facility of ease, effect on the ability of theSelf-assembled monolayer-forming compound to effectively form aSelf-assembled monolayer, and cost. Such a choice is within theknowledge, ability and discretion of one of ordinary skill in the art.

[0165] Alternatively, the functional group may be chosen from a widevariety of compounds or fragments thereof which will render theself-assembled monolayer “biophobic” as that term is defined below.Biophobic self-assembled monolayers are those with a generally lowaffinity for binding, adhering, or adsorbing biological materials suchas, for example, intact cells, fractionated cells, cellular organelles,proteins, lipids, polysaccharides, simple carbohydrates, complexcarbohydrates, and/or nucleic acids. Biophobic functional groups includepolar but uncharged groups including unsaturated hydrocarbons. Aparticularly preferred biophobic functional group is polyethylene glycol(PEG).

[0166] The central portion of the molecules comprising theself-assembled monolayer-forming compound generally includes a spacerfunctionality connecting the functional group selected to bind the tosurface material and the exposed functionality. Alternately, the spacermay essentially comprise the exposed functionality, if no particularfunctional group is selected other than the spacer. Any spacer that doesnot disrupt self-assembled monolayer packing and that allows theself-assembled monolayer layer to be somewhat impermeable to variousreagents such as etching reagents, as described below, in addition toorganic or aqueous environments, is suitable. The spacer may be polar;non-polar; halogenated or, in particular, fluorinated; positivelycharged; negatively charged; or uncharged. For example, a saturated orunsaturated, linear or branched alkyl, aryl, or other hydrocarbon spacermay be used.

[0167] A variety of lengths of the self-assembled monolayer-formingcompound may be employed in the present invention. If two or moredifferent self-assembled monolayer-forming compounds are used in onestamping step, for example if two or more different self-assembledmonolayer-forming compounds are used in the ink, it is oftenadvantageous that these species have similar lengths. However, when atwo or more step process is used in which a first self-assembledmonolayer is provided on a surface and at least a second self-assembledmonolayer is provided on the surface, the various self-assembledmonolayers being continuous or noncontinuous, it may be advantageous insome circumstances to select molecular species for formation of thevarious self-assembled monolayers that have different lengths. Forexample, if the self-assembled monolayer formed by stamping has a firstmolecular length and the self-assembled monolayer subsequentlyderivatized to the surface has a second molecular length greater thanthat of the stamped self-assembled monolayer, a continuousself-assembled monolayer having a plurality of “wells” results. Thesewells are the result of the stamped self-assembled monolayer beingsurrounded by the second self-assembled monolayer having a longer chainlength. Such wells may be advantageously fabricated according to certainembodiments, for example, when it is desirable to add greater lateralstability to particular biological materials, such as cells, which havebeen captured in the wells. Such wells may also form the basis forreaction vessels.

[0168] Additionally, self-assembled monolayers formed on the surfacematerial may be modified after such formation for a variety of purposes.For example, a self-assembled monolayer-forming compound may bedeposited on the surface material in a self-assembled monolayer, thecompound having an exposed functionality including a protecting groupwhich may be removed to effect further modification of theself-assembled monolayer. For example, a photoremovable protecting groupmay be used, the group being advantageously selected such that it may beremoved without disturbance of the self-assembled monolayer of which itis a part. For example, a protective group may be selected from a widevariety of positive light-reactive groups preferably includingnitroaromatic compounds such as o-nitrobenzyl derivatives orbenzylsulfonyl. Photo-removable protective groups are described in, forexample, U.S. Pat. No. 5,143,854, and incorporated herein in itsentirety by reference, as well as an article by Patchomik, JACS, 92,6333 (1970) and Amit et al., JOC, 39, 192, (1974), both of which areincorporated herein by reference in their entireties. Alternately, areactive group may be provided on an exposed portion of a self-assembledmonolayer that may be activated or deactivated by electron beamlithography, x-ray lithography, or any other radiation. Such protectionsand deprotections may aid in chemical or physical modification of anexisting surface-bound self-assembled monolayer, for example inlengthening existing molecular species forming the self-assembledmonolayer. Such modification is described in U.S. Pat. No. 5,143,857referenced above.

[0169] Another preferred method of patterning the self-assembledmonolayer to have an array matching the first layer 150, for example, isthrough soft lithography methods known in the art. Soft lithography hasbeen exploited by George M. Whitesides and is described, by way ofexample, in U.S. Pat. No. 5,976,826 and in PCT WO 01/70389, both ofwhich are herein incorporated by reference in their entireties. Forexample, the first layer 150 having micro-orifices 300 is placed overthe self-assembled monolayer. The first layer makes conformal contactwith support 140 by sealing against the self-assembled monolayer. Amodifying solution is then placed on the first layer and allowed tocontact the self-assembled monolayer surface exposed by themicro-orifices 300. A “modifying” solution is one that modifies the headgroup of the self-assembled monolayer to achieve a desiredcharacteristic or that adds or removes a desired biomolecule to the headgroup. For example, a tether may be added to the exposed self-assembledmonolayers head groups, which in turn captures a protein, which in turnsprovides an affinity for the cell to be patterned subsequently throughthe first layer 150 or the second layer 160.

[0170] Preferred surface portions of the patterned self-assembledmonolayer are cytophilic, that is, adapted to promote cell attachment.Molecular entities creating cytophilic surfaces are well known to thoseof ordinary skill in the art and include antigens, antibodies, celladhesion molecules, extracellular matrix molecules such as laminin,fibronectin, synthetic peptides, carbohydrates and the like.

[0171] In a preferred embodiment of the present invention, theself-assembled monolayers are modified to have “switchable surfaces.”For example, self-assembled monolayers can be designed with a “headgroup” that will capture a desired molecule. The head group is thensubsequently modified at a desired point in time to release the capturedmolecule. In a preferred embodiment of the present invention, the headgroup is modified such that after release of the captured cell, the headgroup no longer will attract and attach subsequent cells. This releaseis important to allow the patterned cells to migrate. If aself-assembled monolayer did not have a “switchable” head group, themigration of the cell may be hindered. An example of a “switchable”control is depicted in FIG. 14. This figure depicts a particularpeptide-presenting compound that allows cells to attach to itself. Uponapplication of an electrical potential, the peptide presenting compoundis cleaved causing the release of cells from the support. Importantly,the portion of the peptide presenting compound that remains afterapplication of the electrical potential is unable to bind cells, andthus eliminates the potential for non-specific cell binding.

[0172] It is also often desirable to utilize a bioinert support materialto resist non-specific adsorption of cells, proteins, or any otherbiological material. The most successful method to confer thisresistance to the adsorption of protein has been to coat the surfacewith poly(ethylene glycol) PEG. A variety of methods, includingadsorption, covalent immobilization, and radiation cross-linking, havebeen used to modify surfaces with PEG. Polymers that comprisecarbohydrate units also passivate surface, but these material are lessstable and less effective than PEG. A widely used strategy is topreadsorb a protein—usually bovine serum albumin—that resists adsorptionof other proteins. In addition, self-assembled monolayers that areprepared from alkanethiols terminated in short oligomers of the ethyleneglycol group [HS(CH₂)₁₁(OCH₂CH₂)_(n)OH:n=2-7] resist the adsorption ofseveral model proteins. Even self-assembled monolayers that contain asmuch as 50% methyl-terminated alkanethiolates, if mixed witholigo(ethylene glycol)-terminated alkanethiolates, resist the adsorptionof protein. Further, self-assembled monolayers that are terminated inoligo(ethylene glycol) groups may have broad usefulness as inertsupports, because a variety of reactive groups can be incorporated inself-assembled monolayers in controlled environments.

[0173] In contrast to using a bioinert treatment or support material, bychoosing an appropriate support or treatment, the surface can bemodified to have any desired functionality. For example, the support canbe treated to have immobilized biomolecules such as other cells,DNA/RNA, chemicals, or other biological or chemical entity. For example,the attachment and spreading of anchorage dependent cells to surfacesare mediated by proteins of the extracellular matrix, e.g. fibronectin,laminin, vitronectin, and collagen. A common strategy for controllingthe attachment of cells to a surface therefore relies on controlling theadsorption of matrix proteins to the surface. Therefore, a preferredcoating 220 includes extracellular matrix proteins, or hydrogels,including matrigel, or other coatings that mimic the extracellularmatrix.

[0174] In another example, the coating comprises an immobilized entitythat may or may not affect the behavior of the cell migration ormotility, such as drugs, toxins, metabolites, test agents, etc. Afterplacing the cells into the orifices of the first layer 150, the cellssettle onto the surface of the support and are thus affected by theimmobilized entity.

[0175] In yet another preferred embodiment, coating 220 may comprisecoatings that provide a more in vivo-like environment for the arrayedcells. Since cells in vivo are usually in contact with other cell types,and since it has been observed that cell to cell contact effects thebehavior of cells, a preferred coating 220 also comprises a secondarycell type to that of the primary cells to be arrayed. For example,cancer cells are surrounded by stromal cells. Thus, to more accuratelycorrelate the migration or movement of cancer cells in vitro with whatoccurs in vivo, it is desirable to provide a coating 220 of stromalcells before patterning the cancer cells onto the coating. The growingof two different cell types together has been coined “co-culture” bythose skilled in the art. Some commonly known co-cultures includehepatocytes/fibroblasts; astrocytes/dendrocytes; endothelialcells/leukocytes; and neural cells/glial cells. The present inventioncontemplate employing co-culture systems by providing a coating of onecell type and then arraying the second type onto the cellular coatedsupport.

[0176] In yet another embodiment of the present invention, the support140 may have a surface treatment in the form of “physical”modifications, such as striations, grooves, channels and indentations toeffect cell motility and migration.

[0177] The cell migration assay plate of the present invention allowsfor a broad range of patterns to be applied. For example, the entiresupport may define a pattern that is uniformly distributed across thesupport. FIG. 6(a) depicts one embodiment of the present inventionwherein the first layer 150 has a plurality of micro-orifices uniformlydistributed across the first layer 150. When the second layer 160 ofFIG. 6(b) is placed onto the first layer 150 shown in FIG. 6(a), thearrangement of micro-wells 300 with macro-wells 170 as shown in FIG.6(c) is created.

[0178]FIG. 7(a) depicts another embodiment of the present invention, inwhich the support 140 is configured by arraying the micro-orifices 300of the first layer 150 into discrete geometric patterns. When the secondlayer 160 of FIG. 7(b) is then placed onto the first layer 150 shown inFIG. 7(a), the arrangement of micro-wells 300 with macro-wells 170 asshown in FIG. 7(c) is created. These discrete areas preferably have thesame size and pitch of standard micro-titer plates. The discrete areasmay contain any desired number of individual patterned cells. Forillustration purposes, FIGS. 7(a) depicts the first layer 150 having 6discrete geometric patterns, each pattern occupying a corresponding areaof the first layer 150. Within each of these 6 discrete areas are 10micro-orifices. After applying cells to the support 140 through themicro-orifices 300, the resulting patterned support 140 will define sixmacro-regions, each of these macro-regions defining ten micro-regions ofpatterned cells. Each micro-region may contain one cell or a pluralityof cells.

[0179] The description of the embodiment of the present invention setforth above with respect to FIGS. 7(a)-7(c) demonstrates the flexibilityof the cell migration assay plate of the present invention. By varyingthe number, size, and pitch of the micro-orifices 300 of the first layer150 and/or macro-orifices 170 of the second layer 160 of assay plate100, any desired configuration or pattern of cells can be achieved.According to the present invention, any number of macro-wells 151 couldbe defined by an assay plate, and in addition, each macro-well couldcircumscribe any number of micro-wells 141 to create a desired geometricpattern. As previously mentioned, preferred embodiments of the presentinvention have discrete areas that—match the number, size and pitch ofthe footprint of standard micro-titer plates used in the industry. Forexample, one preferred embodiment comprises a second layer 160 having 96discrete macro-orifices 170 that match the footprint of a 96-wellmicro-titer plate. Arranged on the first layer 150 so as to be situatedwithin each one of the 96 discrete macro-orifices 170 are, for instance,100 micro-orifices 300 configured to receive solutions of cells. Theresulting arrayed support 140 has 96 areas, each having 100 separatemicro-regions of cell(s).

[0180] In another embodiment of the cell migration assay plate of thepresent invention, there are means for aligning the layers of thedevice. For instance, in order to align the micro-orifices 300 of thefirst layer 150 with the macro-orifices 170 of the second layer 160, thefirst layer 150 may need to be aligned precisely on the second layer160. FIG. 3(a) depicts physical aligning means 190 and visual aligningmeans 192, one or both of which may be employed in the presentinvention. Physical aligning means 190 may comprise protrusions, pins,prongs, or the like that extend from the support. In one embodiment ofthe present invention, physical aligning means 190 are prongs thatprotrude from the support 140 and extend through guidance orifices 194in layers placed thereon. An example of guidance orifices 194 is shownin FIG. 4(a). In another embodiment, the support 140 has a raised outerframe or ridge comprising a wall made of rigid material on the perimeteredge of the support 140, such as wall 196 illustrated in FIG. 3(a). Thespatial constraints of the frame or wall 196 guide layers placed thereoninto the correct position. The visual means 192 may include markings onthe support 140 and/or on other layers to guide the placement of eachadditional layer on top of the next layer. Visual aligning means 192include, but are not limited to, markings such as dots or cross hatches.

[0181]FIG. 8(a) is a top plan view of a cell migration assay plate 100in accordance with still another alternative embodiment of the presentinvention. In this embodiment, rather than being cylindrically shaped,the macro-wells 151 are funnel-shaped. Moreover, rather than being openand exposed to the atmosphere, the macro-wells 151 in this embodimentare shown as being capped with a cap 820. The cap 820 may comprise oneor more materials configured to conform to at least in part an uppersurface of the second layer 160 and sealably engage itself with-theopenings of the macro-wells 151. In the embodiment of the presentinvention shown in FIG. 8(b), cap 820 includes a seal 930 made of afirst material that acts as a plug with respect to the macro-well 151,and a continuous covering layer 835 made of a second material andextending across an upper surface of the second layer 160. The cap 820may be useful for preventing evaporation of assay solutions that may beplaced into the macro-wells 151 and/or during the storage and transportof the cell migration assay plate.

[0182]FIG. 8(a) and 8(b) also depict a lining 835 that may be used toform and line each macro-well 151 of the cell migration assay plate 100.This lining, which has a top edge 836 and bottom edge 837, may be madefrom a material different from the material of the cell migration assayplate 100 in order to provide the macro-wells 151 with properties otherthan those attributable to the cell migration assay plate 100 material.Moreover, the lining 835 may also be used to form the cell migrationassay plate 100 during its manufacture by positioning the linings inspace and then by pouring the material of the cell migration assay platematerial around them.

[0183] The funnel shape of the linings 835 and of their correspondingmacro-wells 151 is seen in FIG. 8(b). The first layer 150, support 140,cap 820, seal 930, top edge 836, and bottom edge 837 can also be seen inthis figure. Moreover, as is also seen in FIG. 8(b), the linings 835define and form the shape of the macro-wells 151 and sealably engage thefirst layer 150 located on top of the support 140.

[0184] FIGS. 8(c) and 8(d) depict another embodiment of a cell migrationassay plate 100 comprising plugs 320. In the shown embodiment, a secondlayer 160 defining macro-orifices 170 is placed onto a support 140.According to one embodiment of the invention, plugs 320 having anoutside diameter “OD” smaller than an inner diameter “ID” of amacro-well 151 is configured for insertion into each macro-orifice 170.In the embodiment shown, the height of the plug, designated as “HP,” isshorter than the depth of the second layer corresponding to a depth ofthe macro-well 151 and, designated as “DW,” so as to enable testsubstances to be added at a subsequent time into the openings of themacro-orifices 170. Each of these plugs 320 has a membrane 350 at abottom surface thereof, membrane 350 defining micro-regions 370 of cellsin a defined geometric pattern. Plugs 320 are-preferably dimensioned soas to be insertable into respective macro-wells 151 of the assay plate100.

[0185] In another embodiment of the cell migration assay plate accordingto the present invention, cap 185 (not shown) is placed on top of thesecond layer 160. Cap 185 may be composed of rigid or flexiblematerials, described previously. Cap 185 is useful for preventingevaporation of assay solutions that will be placed onto the devicethrough the macro-orifices 170.

[0186] Fabrication of the Qualitative Cell Migration Assay Plate

[0187] The cell migration assay plate according to the present inventionhaving a first layer 150 and a second layer 160 may be manufacturedaccording to the present invention by two methods: a single-piecefabrication method on the one hand, and a two-piece fabrication on theother hand, as will be described further below. It is understood,however, that the present invention includes within its scope othermethods for manufacturing the assay plate according to the presentinvention that would be within the knowledge of a person skilled in theart.

[0188] By forming the cell migration assay plate of the presentinvention in a single sequence of pouring, degassing, and curing, themanufacturing cycle time is reduced and a seal between the first layerand the second layer of the device is improved. The main advantage ofthis method is that it requires no manual handling of a preferredmaterial (a thin PDMS membrane). According to various embodiments of theinvention, a single-piece fabrication method may be employed wherein thedevice is formed on an original silicone/photoresist membrane master.FIG. 9(a) depicts a device comprising a silanized array of PDMSmacrocosms 502 to form the macro-orifices in the second layer. Thesemacrocosms 502 are formed by casting PDMS against a standard micro-titerplate 100 as shown in FIG. 9(b), such as a 96-well micro-titer plate,for example, and by sealing the resulting structure to a glass slide504.

[0189] The use of PDMS macrocosms 502 provides a convenient method forfabricating the patterning layers. Silanization of PDMS using aperfluorosilane renders its surface resistant to lesion by the PDMSprecursor and eliminates cross-linking of PDMS into the posts. Themacrocosms are preferably prepared using PDMS, but many other materialsmay be used, such as Teflon, metal (e.g., aluminum), and other polymers.As previously mentioned, a 96-well plates may be used as a master,though lower and higher densities based on 12, 24, 384 and 1586 wellconfigurations may also be used. The standard micro-titer platefootprints are preferred because many detection schemes have beendeveloped for the same. After the PDMS macrocosms 502 are oxidized inair plasma (1 minute at 300 mTorr, 6 watts), they are silanized byimmersion in a fluorosilane solution (1% by volume in methanol).

[0190] FIGS. 10(a) through 10(c) illustrate schematically respectivestages corresponding to one embodiment of a method according to thepresent invention in which the first layer and the second layer of anembodiment of the assay plate of the present invention may be fabricatedusing the macrocosms 502 as previously described. Although the followingdescription is with respect to the embodiments of the assay plate ofFIGS. 1(a)-7(c), it is understood that the methods described withrespect to FIGS. 10(a)-11(c) and with respect to cell patterning areequally applicable to other embodiments of the assay plate of thepresent invention. Prior to the performance of the step shown in FIG.10(a), a PDMS precursor is spin-coated onto a pattern of photoresistposts arranged in an array of any desired shape, diameter and pitch inorder to produce a first layer, such as first layer 150. A preferredarray has 100 μm-diameter posts in a 3″×3″ array (200 μmcenter-to-center period). The first layer 150 is then cured on themaster. Next, as is shown in FIG. 10(a), the silanized macrocosms 502are placed on top of the membrane and a weight 503 (approximately500-1000 g) is placed onto the glass backing 504. The macrocosms 502seal against the first layer 150, i.e., the PDMS membrane. As shown inFIG. 10(b), PDMS prepolymer 505 is poured onto the first layer 150, andflows around the macrocosms 502 and forms a thick (˜5 mm) plate 505 ontop of the first layer 150. As shown in FIG. 10(c), after curing thePDMS, the weight 503 and the macrocosms 502 are removed, and theresulting first layer 150 and second layer 160 together can be peeledoff the master more easily and reproducibly than the first layer 150alone.

[0191] FIGS. 11(a) through 11(c) illustrate schematically respectivestages corresponding to another embodiment of a method of the presentinvention according to which the first layer and the second-layer of anembodiment of the assay plate of the present invention may be fabricatedusing the macrocosms 502 as previously described. The shown method,contrary to that of FIGS. 10(a)-10(c), does not incorporate aspin-coating step. Instead, the first layer, such as first layer 150,and the second layer, such as second layer 160, are formed and curedsimultaneously. Here, as shown in FIG. 11(a), the macrocosms 502 areplaced directly onto a silicon wafer 506 patterned with photoresistposts 506 a. Then, as shown in FIG. 11(b), a PDMS precursor is pouredonto the silicon wafer 506. Then, as shown in FIG. 11(c), a vacuum isapplied to the device so as to remove any air trapped under themacrocosms and to urge the PDMS to fill the spaces between thephotoresist posts 506 a and between the macro-posts 502.

[0192] An alternative fabrication method (not shown) involves theseparate formation of the second layer, such as second layer 160, andthe first layer, such as first layer 150, followed by the assembly, viaadhesion, of the layers. This method is advantageous as it lends itselfwell to high throughput—the first layer and the second layer arerelatively straightforward to make using conventional processes such asspin-coating and molding. After the first layer and the second layer aremade, they are aligned and bonded together. Care must be taken whenhandling the thin membrane component.

[0193] The assembly of the two layers 150 and 160 may be accomplishedusing one of multiple methods, for instance plasma oxidation, using anadhesive layer, using double sided tape or using mechanical methods.When an adhesive layer is used, a PDMS precursor may be used to bond thetwo layers. This precursor may be crosslinked either thermally orphotochemically. Additionally, any other “glue” that can adhere to thePDMS surface may be used. The present invention also contemplates thatdouble-sided adhesive tapes that can adhere strongly to the surface ofPDMS can be used. In various other embodiments, mechanical methods mayalso be employed. In some applications, mechanical pressure may bemaintained on the layers throughout the course of an experiment. BecausePDMS can deform under pressure and act as a “gasket,” mechanical sealingis a practical solution to assembling the components. One of theadvantages to using mechanical methods over glues and tapes is that theassembled structure may be disassembled quickly without resulting in anydamage to the device or the patterned material.

[0194] The present invention is also directed to methods of patterningcells using the cell migration assay plate of the present invention. Ina preferred method of patterning cells according to the presentinvention, a first layer, such as first layer 150, is placed on asupport 140, and the second layer, such as second layer 160, is placedon top of the first layer. The positioning of the macro-orifices 170over the micro-orifices 300 may be assisted by the use of an alignmentmeans as discussed above. In one embodiment, cells are patterned throughthe first layer 150 and allowed to settle and are applied to the support140 to create an arrayed support 140 having micro-regions of adheredcells. Each micro-orifice 300 can receive the same cell containingsolution or a different cell containing solution. The first layer 150may then be removed. In this embodiment, it is preferred that the firstlayer 150 is coated with BSA or other cytophobic materials to resistcellular attachment. The second layer 160 is then aligned over thearrayed support 140. The macro-orifices 170 define macro-wells 151encompassing a plurality of micro-regions of cells. Test agents can thenbe added through the macro-wells 151 to contact the micro-regions ofarrayed cells. Each macro-well 151 can receive the same or a differenttest agent.

[0195] In an alternate embodiment, the first layer 150 is placed on thesupport 140, and the second layer 160 is placed on top of the firstlayer 150. The positioning of the macro-orifices over the micro-orifices300 may be assisted by the use of an alignment means as discussed above.Cells of a first type are patterned through the first layer 150 and areapplied to the support 140 in a pattern to create micro-regions ofadhered cells. The first layer 150 is removed. The second layer 160 isthen mated to the support. The second layer 160 has macro-wells 151 thatencompass a plurality of micro-regions of the first cell type. Asolution having cells of a second type is then placed into each of themacro-wells 151 to fill in around the micro-regions of the first celltype. The cells are allowed to attach to the support. Test agents thenmay be added into the macro-wells to contact the cells on the support140. Each macro-well 151 can receive the same or a different test agent.FIG. 12(a) depicts a first cell type, e.g., MS1 (endothelial cancercells), patterned into micro-orifices 300 of the first layer 150. Afterremoval of the first layer 150, a second type of cells, e.g., 3T3 normalfibroblast cells, as shown in FIG. 12(b) is arrayed around the firstcell type to create an overlayed arrangement as shown in FIG. 12(c). Themethod illustrated in FIGS. 12(a)-(c) is commonly referred to asresulting in a “co-culture.”

[0196] In another embodiment, both the first layer 150 and the secondlayer 160 are brought into contact with each other and are placed on topof the support 140. The cells are patterned through the macro-wells 151of the second layer 160 and through the micro-orifices 300 of the firstlayer 150 to contact and attach to the underlying support 140. Theresulting patterned support 140 has micro-regions of attached cells.Test agents are then added to the macro-wells 151 to contact thepatterned cells. Each macro-well 151 can receive the same or a differenttest agent. In another embodiment, discussed previously above, thesupport 140 is first coated with a coating 220 before the first layer150 is mated to the support and before the micro-orifices receive asolution of cells.

[0197] Because the cells are patterned in predetermined arrays by theirplacement through the micro-orifices 300 of the first layer 150, theexact positions of the cells are known and identifiable. The effects onmovement or migration of the arrayed cells can be studied more preciselyby measuring the movement or lack of movement of the cells away fromtheir starting positions. In addition, since the arraying is broughtabout by the constraints of the micro-orifices 300 of the first layer150, the precise pattern can be duplicated across the support in theareas encompassed by each of the plurality of macro-wells 151 by havingthe same geometric pattern of micro-orifices 300 in each macro-well 151.This reproducibility of cellular patterns on a support 140 provides fora quick and reliable comparison of cellular movement of the cells ineach macro-well 151 against other macro-wells 151. Furthermore, withineach macro-well 151, each micro-region of cell(s) is illustrative of theother micro-regions within that macro-well 151. For example, becauseeach of the micro-orifices 300 can be fabricated to be of the same sizeand shape, and the same amount of cell(s) can added to eachmicro-orifice 300, one can observe a micro-region of cells patterned bya first micro-orifice 300 at a first time point and later observe asecond micro-region of cells patterned by a second micro-orifice 300 ata second time point and compare the observations recorded at the twotime points. Since the cell(s) in each of the micro-orifices 300 wereexposed to the same conditions, and were patterned by identicalmicro-orifices 300, one need not go back to the previously observedmicro-region 300 over the time course of the assay.

[0198] Further, having cells patterned in identical predeterminedstarting positions in each macro-well 151, the effects of a first testagent on a cell population in a first macro-well 151 can more accuratelybe compared to effects of a second test agent on a cell population in asecond macro-well 151.

[0199] The flexibility of the cell migration assay plate of the presentinvention and the flexibility in the methods of patterning cells usingthe cell migration assay plate of the present invention provide fornumerous cell migration assay configurations. A virtually unlimitedamount of configurations can be achieved simply by choosing variousdimensions, numbers, shapes and pitch of micro-orifices 300 andmacro-orifices 170, as well as by modifying the coating 220 on thesupport 140.

[0200] Using the cell migration system and the cell migration assayplate of the present invention, novel cell migration assays can beperformed. These assays measure the migration or motility of patternedcells. Since the present invention provides for patterning cells indiscrete arrays, the measurement of cell movement/migration is moreaccurate as it measures motility or migration away from a predeterminedstarting position created by the micro-orifices of the first layer. Inaddition, the cell migration/motility assays of the present inventionprovide for ongoing/real-time monitoring of the cells as the cells canbe visualized through light or flourescent microscopy and need not bestained and fixed for counting as previously required by the Boydenchambers. The present invention contemplates the monitoring andobservation of cellular movement or migration of numerous cell types,which will provide much needed information about processes in the bodythat occur as a result of cell movement.

[0201] Cellular movement is implicated in numerous systems and responsesin the body. For example, leukocyte movement is involved in inflammatoryand immune responses. Leukocyte cell classes that participate incellular immune responses include lymphocytes, monocytes, neutrophils,eosinophils, and mast cells. Leukocytes accumulate at a site ofinflamation and release their granular contents such as varioushydrolytic enzymes an other toxic components into the extracellularspaces. As a result, the surrounding tissue is damaged. Numerous chronicinflammatory disease are thought to involve the aberrant presence ofleukocytes in tissues. Infiltration of these cells is responsible for awide range of chronic inflammatory and autoimmune diseases, and alsoorgan transplant rejection. These diseases include rheumatoid arthritis,psoriasis contact dermatitis, inflammatory bowel disease, multiplesclerosis, atherosclerosis, sarcoidosis, idiopathic pulmonary fibrobsis,allograft rejection and graft-versus-host disease, to name a few.

[0202] In another process of the body, cancer cells break off from atumor and metastasize to other parts of the body. Thus, cell migrationassays that provide a reliable study on the ability of potential drugcandidates to inhibit cancer cell growth and/or metastasis would providevaluable information to the field of oncology.

[0203] In one embodiment of the cell migration/motility assay of thepresent invention, cells are first allowed to migrate through themicro-orifices 300 of the first layer 150 onto the support 140 toproduce an arrayed support 140. The cells are allowed to attach and growto confluence within the micro-orifices 300. The first layer 150 is thenremoved. The second layer 160 is placed on top of the arrayed support140 to form macro-wells 151 encompassing areas of patterned cells. Atest solution is added through the macro-orifices 170 of the secondlayer 160 and allowed to contact the arrayed cells. The effects of thistest solution on cell movement or migration is then observed. FIGS.13(a) through 13(c) illustrate the stages according to the aboveembodiment of the assay of the present invention. FIG. 13(a) illustratesa second layer 160 sealed to a first layer 150. FIG. 13(b) illustratescells that have been patterned through the first and second layers 150and 160 onto the support 140 and are allowed to grow to confluencewithin the micro-orifices 300. An example of this is shown in FIG.13(d). As shown in FIG. 13(c), the first layer 150 is then removed, andthe cells arrayed on the support 140 are permitted to migrate, anexample of which is shown in FIG. 13(e). The observation can beperformed using any method known in the art, including but not limitedto light microscopy and fluorescent microscopy.

[0204] In another embodiment of the cell migration/motility assay of thepresent invention used in conjunction with embodiments shown in FIGS.1(a)-7(c), the support 140 is treated directly with test agents orcoated with a membrane having test agents coated thereon. The agents arethen tested to determine whether they exert any chemotactic effect. Insuch a scenario, the micro-orifices 300 of the first layer 150 aresmaller in diameter than the size of an individual cell to be plated.The cells are plated and allowed to squeeze through pre-defined arraysof micro-orifices 300 in response to the chemotactic agent on thesupport 140. The support 140 or the membrane is then observed for thecells. Since the micro-orifices 300 are designed in a predeterminedgeometric pattern, the analysis and determination of cell migrationthrough the first layer 150 onto the support results from a quick visualinspection of the support 140 for cells. For example, if themicro-orifices are arrayed in a 10×10 pattern (for a total of 100cells), a quick visual review of the support or membrane would informthe scientist what percentage of cells migrated through themicroorifices. A high percentage of cells migrating corresponds to astrong chemotactic substance and a low percentage corresponds to a weakchemoattractant. In contrast to transwell chemotactic assays thatinvolve establishing a top and bottom base line, no base linemeasurements are needed for the above assay to analyze the strength orweakness of a chemotactic substance.

[0205] In another embodiment of the cell migration/motility assay of thepresent invention, the support, such as support 140, is first coatedwith a coating 220 such as extracellular matrix proteins or matrigel(not shown). Cells are then plated onto the coated support. Themigration or movement of the cells through the matrigel is observed. Instill another embodiment of the assay of the present invention, thematrigel can contain test agents.

[0206] The cell migration/motility assay of the present invention allowsone to study the effect of test agents and others both on cell motilityand on cell shape. For example, cells may be patterned throughmicro-orifices, such as micro-orifices 300, of the first layer 150. Thecells are allowed to attach to the support 140 and to grow toconfluence. The walls of the micro-orifice 300 constrain the cell(s) andthe cells take on the shape of the micro-orifice 300, e.g., circular. Atest agent is applied through the micro-orifices 300 and is allowed tocontact the cells. The first layer 150 is removed and the cells areobserved. If the test agent affects cell movement, the cell will be“stuck” in place as it was patterned and may not change shape, i.e., itwill remain circular if the patterning member had circular orifices. Onthe other hand, if the test agent does not effect cell movement, thecell will move away from its original patterned position and changeshape from the patterned circular shape since the constraints of thefirst layer 150 had been removed. FIG. 15(a) illustrates, in its leftcolumn, an example wherein control cells, at various time intervals,e.g., 2 hours and 5 hours, are shown to have migrated away from theiroriginal pattern, designated as “hr 0.” In contrast, cells treated witha common cancer drug, taxol, have retained their original circularpattern after these same time intervals, as shown in the right column ofFIG. 15(a). FIG. 15(b) is a graph of the effect of variousconcentrations of taxol on cell movement as performed by a cellmigration/motility assay of the present invention.

[0207] Alternatively, the test agent can be added before the cells havegrown to confluency, i.e. the test agent is added to the cells beforebeing patterned through the micro-orifices 300. If the test agent has noeffect on cell motility, the cells will spread and achieve the shape ofthe micro-orifice 300. In FIGS. 16 and 17, a micro-orifice 300 iscircular in shape. In the left column of FIGS. 16 and 17 are the controlpanel, which illustrate the cells having grown to a confluent circularpattern. As shown in the remaining columns, the cells that were treatedwith various test agents (nocodazole, colchicine, vinblastine, andpaclitaxel) had their cellular movement arrested and thus never achieveda circular confluent pattern.

[0208] One embodiment of the present invention allows one to study theeffect of test agents on cell proliferation as well as cell movement.This is particularly useful in cancer studies where proliferation ratesare high. In this embodiment, the cells to be patterned are preferablystained or fluorescently tagged with two different stains or tags: thenuclei are stained with a different dye or fluorescent tag than the restof the cell (i.e. a cytoplasmic dye or tag). The cells are patternedthrough the micro-orifices 300 of first layer 150. The cells are allowedto attach to the support 140 and to grow to confluence. A test agent isapplied through the micro-orifices 300 and allowed to contact the cells.Alternatively, the test agent is added to the cells before the cells arepatterned through the micro-orifices. The first layer 150 is removed.The cells are then observed for migration or movement and/orproliferation. Using two different tags or dyes, allows for theobservation and recordation of cell number and increase thereof, and/orcell movement. Using this information in combination allows one todeconvolute the effect of motility from proliferation. That is, when thecells are later observed, having moved away from the original pattern,one can determine wether it is because of cell movement alone,proliferation alone, or the combination of movement and proliferation,by simply counting and comparing the number of nuclei at some laterpoint in time compared to the number of nuclei at the beginning of theassay, i.e. at time zero.

[0209]FIG. 18 demonstrates that the assays of the present invention canmeasure cell movement and are not merely measuring cell division. Overtime the cells are seen to spread/move away form their original pattern,but their number remains essentially constant.

[0210] The present invention also includes methods of identifyingmicrobes, methods of screening for the activity of drugs, methods fordetecting toxic substances and methods for detecting intercellularreactions. In these various methods, solutions or suspensions containingthe desired cell affecting agent are flowed in intimate contact with theliving cells through the macrowells/macro-orifices. The effect(s) of thecell affecting agent on cell motion or migration is then monitored andmeasured.

[0211] The present invention may be used with a wide variety ofprokaryotic and/or eukaryotic cells. Examples of such cells include, butare not limited to, human keratinocytes, murine L fibroblastic cells,canine MDCK epithelial cells, hamster BHK fibroblastic cells, murineCTLL lymphocyte cells, tumor cells and bacteria. In general, any livingcells, including transfected cells, that can be successfully patternedmay be used. The cells may be labeled with fluorescent markers known inthe art, such as fluorescein, to assist in microscopic viewing.

[0212] Cell affecting agents can be anything that affects cell motilityor migration. Examples of cell affecting agents include, but are notlimited to, irritants, drugs, toxins, other cells, receptor ligands,receptor agonists, immunological agents, viruses, pathogens, pyrogens,and hormones. Examples of such cell affecting agents further includeirritants such as benzalkonium chloride, propylene glycol, methanol,acetone, sodium dodecyl sulfate, hydrogen peroxide, 1-butanol, ethanol,and dimethylsulfoxide, drugs such as valinomycin, doxorubicin,vincristine, ribavirin, amiloride and theophylline; hormones such at T₃and T₄, epinephrine and vasopressin; toxins such as cyanide,carbonylcyanide chlorophenylhydiazone, endotoxins and bacteriallipopolysaccharides; immunological agents such as inter-leukin-2,epidermal growth factor and monoclonal antibodies; receptor agonistssuch as isoproterenol, carbachol, prostaglandin E₁ and atropine; andvarious other types of cell affecting agents such as phorbol myristateacetate, magnesium chloride, other cells, receptor ligands, viruses,pathogens and pyrogens. In addition, the present invention can also testthe synergistic effect that some of the cell affecting agents may haveon other agents. In other words, the test agents maybe combined andmixed as necessary to better understand their combined synergisticproperties.

[0213] In one cell migration/motility assay of the present invention,cells are patterned onto the support 140 through the micro-orifices 300of the first layer 150. The cells are grown to a certain cell cyclestage and arrested in that stage of cell growth. Test agents are thenadded to the patterned cells and the effects of the agents are observedand monitored. The same test agent may be applied to the same cells atdifferent life cycle stages and compared against each other to shedlight on the effect of the test agent at different points along the cellcycle. In another embodiment, cells are “captured” at a certain cellstage by incubating them elsewhere but capturing them on a supporthaving a coating of a ligand that would “grab” a cellular “tag” such asa protein, that is expressed only at a specific desired cell life cycle(e.g. G1, S, G2, M(standard cell cycle) or S, M(early embryonic cellcycle). In such an embodiment, the coated support would capture onlythose cells at desired life cycle stage.

[0214] In the qualitative cell migration system of the presentinvention, such as system 190 shown in FIG. 1(a), the observation system110 and the controller 120 may be used to observe and analyze thereal-time movement and behavior of cells as they respond to differentand various stimuli. The observation system 110 and controller 120 mayprovide for real-time observation via a monitor (which is not shown).They may also provide for subsequent playback via a recording systemeither integrated with these components or coupled to them. In eithercase, these components may also monitor and analyze the cells as theyprogress through their reaction to the stimulus. System 190 may includeany suitable observation system and controller as would be within theknowledge of a person skilled in the art.

[0215] The observation system 110 may include a microscope, high-speedvideo camera, or high-resolution digital camera, and/or an array ofvideo cameras, and an array of individual sensors. Standard opticalmicroscopy techniques can be used in a parallel setup to quantify themigration. Preferably, an inverted light field phase contrast microscopecan be used to view the live cells. The observation system is connectedto a controller to receive input for various observation parameters. Thedata observed by the observation system is sent to the controller forprocessing in a conventional manner.

[0216] Each of these embodiments allow the monitoring of the movementand behavior of the cells before, during, and after the stimuli,reactant or other test compound is introduced. At the same time, theobservation system 110 may also generate signals for the controller 120to interpret and analyze. This analysis can include determining thephysical movement of the cells over time as well as their change inshape, activity level or any other observable characteristic. In eachinstance, the conduct of the cells being studied may be observed inreal-time, at a later time or both.

[0217]FIG. 19 is a schematic diagram of a system for measuring themigration or motility of cells, in accordance with one embodiment of thepresent invention. The system may use an inverted microscope 1 as shownin FIG. 19, which uses standard objectives with magnification of1-100×to the camera, and a white light source (e.g. 100 W mercury-arclamp or 75W xenon lamp) with power supply 2. In alternate embodiments,the system may use an upright microscope. The system also includes an XYstage 3 to move the qualitative cell migration assay plate 4 in the XYdirection over the microscope objective. A Z-axis focus drive 5 movesthe objective in the Z direction for focusing. A joystick 6 provides formanual movement of the stage in the XYZ direction. A high resolutiondigital camera 7 acquires images from each well or location on thequalitative cell migration assay plate 4. A camera power supply 8provides power to the camera 7. An automation controller 9 controls theautomated aspects of the observation system, and is coupled to a centralprocessing unit 10. A PC 11 provides a display 12 and has associatedsoftware, as is described briefly below. A printer 13 prints datacorresponding to the observed cell migration/motility. Microscopeoculars 14 are positioned so as to be looked through by a user of thesystem.

[0218] In a preferred embodiment of the present invention, theobservation and control systems may be automated and motorized toacquire images automatically. In one embodiment, at the start of anautomated scan, the operator enters assay parameters corresponding tothe sample to be observed and to the arrangement of the qualitative cellmigration assay plate. Assay parameters can include variables such ascell type, number of cells to be patterned into each micro-orifice,shape and pitch of micro-orifices, shape and pitch of macro-orifices,time periods between each image capture (scan), number of images tocapture per macro-well and per scan, etc. Other parameters may includefilter settings and fluorescent channels to match biological labelsbeing used, etc. The camera settings may be adjusted to match the samplebrightness. These parameters are advantageously stored in the system'sdatabase for easy retrieval for each automated run. The user specifieswhich portion of the assay plate the system will scan and how manyfields in each macrowells to analyze on each plate. Depending on thesetup mode selected by the user at step, the system either automaticallypre-focuses the region of the plate to be scanned using an autofocusprocedure to “find focus” of the plate or the user interactivelypre-focuses the scanning region.

[0219] During an automated scan, the software dynamically displays thestatus of a scan in progress, such as by displaying data correspondingto the number of fields in macrowells that have been analyzed, thecurrent macrowell that is being analyzed, and images of each independentwavelength as they are acquired, and the result of the screen for eachmacrowell as it is acquired. The assay plate may be scanned in anynumber of scanning patterns such as top to bottom, left to right, or ina serpentine style as the software automatically moves the motorizedmicroscope XY stage 3 from macrowell to macrowell within the device.Those skilled in the programming art will recognize how to adaptsoftware for scanning of standard microplate formats such as 24, 48, 96,and 384 well plates. The scan pattern of the entire plate as well as thescan pattern of fields within each well are programmed. The systemadjusts sample focus with an autofocus procedure 104 through the Z axisfocus drive 5, and optionally controls filter selection via a motorizedfilter wheel 19 and acquires and analyzes images.

[0220] Automatic focusing algorithms are described in the prior art inHarms et al. in Cytometry 5 (1984), p. 236-243, Groen et al. inCytometry 6 (1985), p. 81-91, and Firestone et al. in Cytometry 12(1991), p. 195-206, which is incorporated by reference herein in itsentirety. U.S. Pat. No. 5,989,835 describes a variation on the abovemethods, which is incorporated by reference herein in its entirety. Theautofocus procedure is called at a user-selected frequency, typicallyfor the first field in the first macrowell and then once every 4 to 5fields within each macrowell. The autofocus procedure calculates thestarting Z-axis point by interpolating from the pre-calculated planefocal model. Starting a programmable distance above or below this setpoint, the procedure moves the mechanical Z-axis through a number ofdifferent positions, acquires an image at each, and finds the maximum ofa calculated focus score that estimates the contrast of each image. TheZ position of the image with the maximum focus score determines the bestfocus for a particular field.

[0221] Because the locations and geometric patterns of the micro-regionsand the macro regions are predetermined, the system can be designed orprogrammed to scan the plate at those locations. The migration ormotility of a cell may be detected by any of a variety of known methodsin the art, including visual monitoring, fluorescence orspectrophotometric assays based upon binding of fluorescently labeledantibodies or other ligands, cell size or morphology, or by the cells'spectrophotometric transmission, reflection or absorptioncharacteristics either with or without biological staining. Standardlight or electron microscopy can also be employed. When the detectionsystem is a microscope, it may be positioned either above or below theassay plate. In the case of fluorescence assays, a detector unit may beplaced above the assay plate or, if the assay plate is translucent,below the assay plate. In the case of transmission spectrophotometricassays, a translucent assay plate is used, a source of electromagneticradiation is placed on one side of the assay plate and a detector uniton the other. In addition to visual monitoring, physical monitoring mayalso be employed. For example, movement of the cells may contactdetectors placed on the assay plate causing changes in the detectors,which can be received and analyzed by the CPU. Because of the smalldistances between individual isolated cells permitted by the presentinvention, detectors employing fiber optics are particularly preferred.Such sources of electromagnetic radiation and such detectors forelectromagnetic transmission, reflection or emission are known in theapplicable art and are readily adaptable for use with the inventiondisclosed herein.

[0222] When an automated detector unit is employed, a standard orcontrol plate may also be provided. Such an assay plate would containmicro-regions including micro-regions to which the cells have notmigrated so that a reference would be provided and the detector wouldrecognize such micro-regions. In addition, micro-regions bearing cellsof known types could be provided to act as references to allow thedetector unit to classify the cells on a subject assay plate.Furthermore, depending upon the nature of the support or treatment onthe support which is chosen, cells of different types may adhere to theassay plate with differing affinities. Thus, depending upon the cells tobe studied and the nature of the support or coatings, a standardcytometric method may be employed on a sample first and then the assayplate and method of the present invention may be employed on the same ora substantially similar sample to calibrate the system.

[0223] For acquisition of images, the camera's exposure time may beseparately adjusted. If the cells are labeled with fluorescent dye, theexposure time is adjusted for each dye to ensure a high-quality imagefrom each channel. Software procedures can be called, at the user'soption, to correct for registration shifts between wavelengths byaccounting for linear (X and Y) shifts between wavelengths before makingany further measurements. The electronic shutter of the camera iscontrolled so that sample photo-bleaching is kept to a minimum.Background shading and uneven illumination can also be corrected by thesoftware using algorithms known in the art.

[0224]FIG. 31 illustrates a method 1100 for testing cellular materialaccording to an embodiment of the present invention. According to themethod, cellular material may be provided in a test bed that initiallydefines a constraint that imposes physical limitations to migration andgrowth of the material (block 1110). A testing agent may be applied tothe cellular material and the constraint may be removed (blocks 1120,1130). Thereafter, the cellular material may be imaged periodically(block 1140). Resultant image data may be compared over time to measureparameters to be captured under test (block 1150). The parameters, asnoted, may include cellular growth, cellular multiplication or cellularmigration under influence of the reactant.

[0225] For example, cells may be patterned through micro-orifices, suchas micro-orifices 1300, of the first layer 1150. The cells are allowedto attach to the support 1140 and grow to confluence. The walls of themicro-orifice 1300 constrain the cell(s) and the cells take on the shapeof the micro-orifice 1300, e.g. circular. A test agent is appliedthrough the micro-orifices 1300 and is allowed to contact the cells. Thefirst layer 1150 is removed and the cells are observed

[0226] Embodiments of the present invention provide image acquisitionand analysis processing for use in connection with the foregoing methodand apparatus. During one or more stages of testing, imaging apparatusmay capture image data of the test apparatus and cellular materialtherein. As noted the captured image data may represent fluorescentcellular material, stained nuclear material or both among other imagecontent contributed by background objects or noise. Image processingstages may analyze the contents of the captured image data to identifygroups of cells, also referred to as “islands,” within the testapparatus. From the identified islands, multiple measurements may becalculated to evaluate parameters such as movement (cell motility),reproduction or multiplication (cell proliferation), growth (cellspreading), shrinking or decrease in size (cell rounding), or celldeath.

[0227] According to an embodiment of the present invention, acquisitionof islands from within image data may occur according to a coarseacquisition stage and a fine acquisition stage. The image acquisitionphase attempts to identify islands and individual cells within an islandthroughout the test apparatus.

[0228]FIG. 34 illustrates a method 1400 of performing coarse islandacquisition according to an embodiment of the present invention. Themethod may begin from captured image data (block 1410) in which themicro-orifices 1210 are oriented with respect to horizontal and verticalaxes of the image data. From the image data, the method 1400 may attemptto identify island rows and columns of micro-orifices at a coarsegranularity (blocks 1420, 1430). Identification of island rows may occurby creating a histogram of image data energy along a first axis of theimage data (say, a vertical axis) (block 1440). From the histogram,coarse island locations may be identified (block 1450) and islandboundaries may be marked between the islands (block 1460).

[0229]FIG. 35 illustrates exemplary image data 1510 (created within theconstraints of the draftsperson's graphics application) and a histogram1520 that may be created therefrom. FIG. 35 illustrates two alternateapproaches to the identification of island locations. In the firstapproach, shown with reference to rows 1 and 2 of micro-orifices, islandcenters may be identified from relative maxima 1530, 1540 of thehistogram. The maxima 1530, 1540 may be taken as coinciding with thecenter of respective rows of micro-orifices. Island boundaries 1550,1560 may be taken as the midpoints between these calculated row centers.Alternatively, when it is known, for example, that micro-orifices occurwith a predetermined spatial distance between rows, row centers may begenerated from a calculation that considers both the histogram maxima1530, 1540 and the predetermined row spacing, such as a least squaresfit

[0230]FIG. 35 illustrates a second approach for detecting islandpositions from the histogram 1520 in connection with rows 3 and 4 of theimage data. In this approach, coarse island locations may be generatedfrom a threshold-tested histogram 1520. A predetermined energy threshold1570 may be applied to the histogram and all vertical regions for whichthe histogram exceeds the threshold may be assigned to respectiveislands. A midpoint 1580 between adjacent region boundaries 1590 may betaken as a dividing line between rows of islands. Again, wherepredetermined geometric relationships between the micro-orifices areknown, such as micro-orifice spacing, midpoint 1580 information maybeintegrated into a larger calculus with the geometric information toidentify island locations.

[0231] Returning to FIG. 34, the method 1400 may identify island columnsfrom the image data as well (block 1430). From a set of image data, themethod 1400 may create another histogram of signal strength, taken alonganother axis of the image data (say, a horizontal axis) (block 1470).The method may identify coarse island locations from the histogram(block 1480) and, thereafter, mark island boundaries to be between theisland locations (block 1490).

[0232]FIGS. 36 and 37 illustrate operation of the column identificationperformed with respect to the exemplary image data of FIG. 35. Incertain embodiments, it may be expected that individual islands will notcoincide with each other in predetermined columns. Thus, wheremicro-orifices are deployed according to a staggered layout, such asthat shown in FIG. 33, or some other layout, it may be appropriate toperform the column identification individually on subsets of image datarather than the entirety of the image data. Thus, FIGS. 36 and 37illustrate operation of the column identification performed respectivelyon odd numbered and even numbered rows of the captured image data. FIG.36 illustrates operation of the column identification process withthreshold-testing of the histogram defines island regions. FIG. 37illustrates operation of the column identification where histogrammaxima are used to identify column centers. These operations may beperformed in a manner that is similar to the row identificationdescribed above.

[0233] The row and column identification processes 1420, 1430 generatedividing lines between respective rows and columns of islands in theimage data. These dividing lines may be used to identify boxes thatdefine a boundary (herein, “bounding boxes”) for each island of cells inthe captured image data. Shown with respect to FIG. 36, dividing lines1610, 1620, 1630 and 1640 define a bounding box for an island 1650 ofcells. Captured image data of each bounding box may be further analyzedin a fine analysis process, described below.

[0234] The foregoing processing may be performed on captured image dataof almost any format. Conventionally, image data occurs asblack-and-white image data, grayscale image data or color image data. Inthe case of black-and-white image data, typically each pixel is assigneda single bit value (either 0 or 1). Grayscale image data typicallyrepresents each pixel by a multi-bit value, such as an eight bit valuewhich would permit 256 quantization levels to be assigned to each pixel.In either of the foregoing cases, the histograms described above may becreated simply by summing the pixel values along each axis. For example,if a histogram is to be created along a vertical axis of the image data,the summing may occur along each pixel row to generate a histogram valueat a corresponding position along the vertical axis.

[0235] Color image data typically includes separate values for eachcolor component at each pixel. Thus, a pixel may have a red colorcomponent, a green color component and a blue color component. Analternative color system may represent image information as a luminancecolor component and a pair of chrominance color components. Histogramsmay be generated by summing a predetermined one of the color components,by summing all of the color components or by calculating an “energyvalue” of each pixel from the components and summing the calculatedenergy values.

[0236] A fine island identification process may follow the coarseidentification process described above. FIG. 38 illustrates a method1800 of identifying islands from image data of bounding boxes, such asthe bounding boxes described from the foregoing embodiments. The method1800 may begin by defining a dilation kernel dimensioned according to apredetermined expectation distance (box 1810). The expectation distancemay be an arbitrary distance chosen by an operator and is related to thesize of the identified island. The operator may alter the expectationdistance at any time to achieve a larger or smaller identified island asdesired. For example, the expectation distance may define the radius ordiameter of the dilation kernel. The method 1800 also may initializedata of a “dilated image” (box 1820). Thereafter, the method mayconsider each pixel in the bounding box. For each pixel, the methodconsiders the captured image data that falls within a dilation kernelcentered at the current pixel (box 1830). If the image data of thedilation kernel indicates that cellular material is represented therein,the method 1800 may set pixels occupied by the dilation kernel in thedilated image data (boxes 1840, 1850). Thereafter or if the image dataindicates that no cellular material is represented therein, the methodmay advance to the next pixel (box 1860).

[0237] The method 1800 generates a second image from the captured data,a dilated image. Once all pixels have been considered, the resultingimage, referred to as the dilated image, may contain one or more dilatedislands.

[0238] According to an embodiment, the methods of FIGS. 34 and 38 may beapplied serially to captured image data to provide both coarse and fineisland acquisition. Of course, other implementations are possible. Inthe circumstance where an image represents a test to be run on a singlemicro-orifice, it would not be necessary to perform the method of FIG.34 to identify bounding boxes of coarse locations of islands.

[0239] In an embodiment, it is not necessary for the method to iterateover every pixel of a bounding box. The method may consider pixelshaving predetermined spacing from each other (e.g., every other pixel,every third pixel, etc.) in each direction. The method need not considerpixels on the outer boundaries of the bounding boxes over which ittraverses.

[0240] The foregoing description of the island acquisition method 1800introduces the concept of an expectation distance. Generally, theexpectation distance is related to a maximum expected distance ofseparation that may occur between any two pairs of cells for which it isappropriate to consider the cells as part of the same “island.”Typically, the expectation distance may be derived from the biologicaltest to be run and may depend on cell types, number of cells, amount andtype of test agents and other factors that are known to influence thebiological properties being measured. Therefore, it may be set on acase-by-case basis.

[0241] As in the foregoing methods, the method of FIG. 38 findsapplication with various types of image data and may be used withvarying levels of sensitivity. For example, in black-and-white data,white pixels (those having values of 1) may represent the presence ofcellular material. Thus, the determination of whether dilation kerneldata represents cellular material may be answered affirmatively if evena single pixel had a value of one. Similarly, in grayscale data,cellular material may be identified for a pixel having a predeterminedvalue (say, half scale or greater—a value of 127 in an 8 bit word).Again, if the value of any pixel exceeds the threshold value, the methodwould be permitted to indicate that cellular material is presenttherein. For color image data, similar calculations may be made. Sincecellular material fluoresces at a predetermined wavelength, it may bepossible to examine only predetermined color components of the imagedata to determine if cellular material is present in a dilation kernel.

[0242] FIGS. 35-37 illustrate operation of the image processing methodsupon idealized data in which cellular material is confined to themicro-orifices. While such presentation is useful to explain operationof the methods, the methods are most useful when applied to image datathat captures cellular migration, spreading, proliferation, rounding, ordeath. FIGS. 39 and 40 are screen shots illustrating operation of theforegoing methods upon image data in which cellular material has beenpermitted to migrate without limitation. FIG. 39 illustrates exemplarysource data and bounding boxes identified from operation of the method1400 of FIG. 34. FIG. 40 illustrates exemplary dilated image datadeveloped from the source image data of FIG. 39. FIG. 40 alsoillustrates island that may have been recognized from the source imagedata. They are circumscribed by bounding boxes of their own.

[0243] Having identified islands of cellular material, the imageacquisition process may generate several independent measurements of theislands that may provide statistically useful biological information,and more preferably, information related to one or more of cellmotility, cell growth, cell proliferation, cell rounding or cell death.

[0244] Additionally, where more than one cell type is deposited in themicro-orifice, the measurements may be calculated based on one type ofcell within a population of mixed cell types rather than on the entirepopulation of cells. These measurements may be calculated for each typeof cell within the micro-orifice and then may be compared to each otherto produce relevant biological information.

[0245] For example, prior to deposition in the micro-orifice, each celltype may be treated with an appropriate label, tag, stain, or dye so asto distinguish and identify each cell type within the samemicro-orifice.

[0246] Such information may include, for example:

[0247] Pixelated cellular area;

[0248] Dilated cellular area;

[0249] Vertical and horizontal lengths;

[0250] Average minimum distance between cells;

[0251] Average distance between cells;

[0252] First polar moment of inertia taken about the island centroid;

[0253] Second polar moment of inertia taken about the island centroid;and

[0254] First and/or second polar moments of inertia taken about theisland centroid, normalized to cellular area.

[0255] Each measurement is discussed in turn.

[0256] The pixelated cellular area calculus counts from source imagedata the number of pixels in a given island that represents the presenceof cellular material. The dilated cellular area counts from dilatedimage data the number of pixels in a dilated island that represents thepresence of cellular material. The vertical and horizontal lengthscalculus respectively represents the height and width of a bounding boxthat surrounds an island or dilated island of cellular material; thesedimensions may be taken from the source image data or dilated image dataof an island as desired.

[0257] The method also may capture the average minimum distance betweencells and the average distance between cells. For these measurements, itmay be useful to identify cell nuclei and compute distances betweenthem. For example, cells may have a nuclear staining agent applied tothem in addition to a fluorescing agent. Captured image data then maycapture not only the cytoplasm as a fluorescent material but they alsomay captured cell nuclei as a predetermined color that may bedistinguishable from the fluorescence within the image. It may be usefulto capture two images of the cells, a first image to capture thefluorescent material and a second image to capture the cell nuclei. Ineither embodiment, cell nuclei may be identified and distinguished fromother artifacts within a captured image.

[0258] The distance parameters may consider the positions of variouscell nuclei in a given island. To compute the average distance betweencells within a given population of cells, the distances between thenuclei of each pair of cells within the population are summed and thesum then divided by the number of unique pairs in the population. Theimage may contain different populations of cells which may include thepopulation of cells defined by a single island within a bounding box,the population of cells defined by two or more islands within a boundingbox or the population of cells defined by the bounding box.

[0259] The average minimum distance between cells also considers thedistance between a given cell and all others in an island. The minimumof these distances is logged. The process may repeat for all other cellsin an island until a set of minimum distances is identified, one foreach cell. Thereafter, these minimum distance values may be averaged todetermine the average minimum distance between cells.

[0260] The first polar moment of inertia considers an island's centroidand the distance of cellular material from this centroid. It involves acomputation of the island centroid and a measurement of each pixelrepresenting cellular material from this centroid. Thereafter, oneintegrates the sums of vector distances from the centroid to each ofthese pixels and multiplies by the area of the island squared.

[0261] The second polar moment of inertia also involves a computation ofthe island's centroid and a measurement of each pixel representingcellular material from this centroid. Thereafter, an integration of thesums of the squares of the vector distances from the centroid to eachpixel may be applied.

[0262] Either the first or second polar moment of inertia calculationsmay be made from the source image data or dilated image data of anisland.

[0263] Additionally, calculations of the first or second polar momentsof inertia may be normalized to the island's area. If the source imagedata is used for the polar moment of inertia calculations, thennaturally the island's source image area can be used for normalization.Similarly, if the dilated image data is used for the inertiacalculations, the island's dilated image area can be used fornormalization.

[0264] The foregoing measurements may, alone or in conjunction with atleast one other measurement, provide biologically relevant information,and more preferably, information related to cell motility, cell growth,cell proliferation, cell rounding, or cell death.

[0265] For example, an increase in pixelated cell area may be indicativeof cell spreading and/or cell proliferation. As another example, adecrease in pixelated cell area may be indicative of cell rounding orcell death.

[0266] An increase in dilated cell area may indicate one or more of cellproliferation, cell spreading or cell motility. A decrease in dilatedcell area may indicate cell rounding or cell death.

[0267] A change in the horizontal or vertical lengths of the boundingbox or change in the average distance between cells or a change in theaverage minimum distance between cells may indicate one or more of cellmotility, cell proliferation, cell spreading, cell rounding or celldeath.

[0268] The relative levels of each of these (e.g., cell spreading, cellrounding, etc.) can be measured more easily by combining cytoplasmic andnuclear stains. For example, when the cells are stained with a nuclearstain, individual cell nuclei may be distinguished from other cellnuclei within a population of cells, thus allowing the number ofdetected nuclei to be summed. The number of detected nuclei may directlycorrelate to the number of cells in a population. When quantified atdifferent time points, the change in the number of cell nuclei detectedover time may be calculated, which may directly correlate to the changein the number of cells in a population over time. A nuclear stain usedin conjunction with a cytoplasmic stain may possibly elucidate the causeof a change in an aforementioned measurement. For example, it may bedetermined that there is no change in the number of cell nuclei detectedand thus, no change in the number of cells in a population over time.One may conclude that a change in a measurement (e.g., pixelated cellarea) is due primarily or wholly to a parameter other than cellproliferation or cell death.

[0269] After a scan of a plate is complete, images and data can bereviewed with the system's image review, data review, and summary reviewfacilities. All images, data, and settings from a scan are archived inthe system's database for later review. Users can review the images ofthe area of the plate analyzed by the system with an interactive imagereview procedure. The digital images produced by the camera are storedin the computer.

[0270] The user can review data using a combination of interactivegraphs, a data spreadsheet of features measured, and images of the areaof the assay plate of interest with the interactive data reviewprocedure. See FIG. 41. Graphical plotting capabilities are provided inwhich data can be analyzed via interactive graphs such as histograms andscatter plots. Users can review summary data that are accumulated andsummarized for all cells within each micro-region with an interactivemicro-region-by-micro-region. Hard copies of graphs and images can beprinted on a wide range of standard printers. All images and data may bestored in the system's database for archival and retrieval or forinterface with a network laboratory management information system. Datacan also be exported to other third-party statistical packages totabulate results and generate other reports.

[0271] As a final phase of a complete scan, reports can be generated onone or more statistics of features measured. Multiple reports can begenerated on many statistics and be printed. Reports can be previewedfor placement and data before being printed.

EXAMPLES Example 1 Procedure for Cell Migration Assay Plate Fabrication

[0272] A topographically patterned master having a plurality of posts isprepared from a photolithographic mask. These posts are elevatedapproximately 100 μm above the background. In one embodiment, thepattern is made up of 24 micro-regions, each containing a circular arrayof 200 μm posts spaced on a 500 μm center. Alternately, instead ofhaving discrete regions of posts, the entire surface of the master maycontain posts. In one preferred embodiment, the master is made ofphotoresist patterned on a 150 mm silicon wafer. To prepare this master,SU-850n photoresist spun at 1300 rpm was used and processed according tothe supplier's specifications.

[0273] A two-component poly(dimethylsiloxane) (PDMS) prepolymer (GelestOptical Encapsulant 41) was mixed and degassed under vacuum before it isspun onto the master. This spin coating was done at a speed high enoughto produce a polymeric membrane (i.e., the thickness of the resultingPDMS film is less than that of the elevated features on the master). Theprepolymer was spun at 2250 rpm for 40 seconds. A rigid frame with thestandard microtiter footprint was then placed around the outer perimeterof the membrane. The master/membrane/frame was then placed on a hotplate109 and the PDMS was cured for seven minutes at 95° C.

[0274] After cooling the master to room temperature, a group of 24 rigidplastic rings was “inked” in thin film of liquid PDMS. The rings werethen placed around the post arrays on the master and the entire assemblywas again heated on a hotplate 100 for two minutes at 95° C.

[0275] The final fabrication step involved filling the area between therings with PDMS to make up the bulk of the device. Here, the PDMS wasinjected via syringe into the space between the rings. The PDMS “ink” onthe rings, which had been partially cured by this point, preventedleakage of PDMS into the membrane regions. The master was again placedon 95° C. hotplate 100 and the PDMS was cured for 30 minutes.

[0276] To remove the cured device from the master, the top surface wasfirst covered with a thin layer of ethanol, which quickly wetted thePDMS. A dull knife was used to cut the interface between the inside ofthe frame and the polymer, which allowed the frame to be removed fromthe master. While the device may be removed with the frame intact (i.e.the frame becomes part of the final device), in this example the framewas used for molding purposes only.

[0277] The device was then covered again with a thin layer of ethanol(to prevent sticking) and manually peeled from the master. Upon removal,the device was rinsed one final time with ethanol before it was driedwith nitrogen gas and placed in a 65° C. oven for solvent evaporation.The device was then stored in a polystyrene dish, which can optionallybe used as the support for studying cell motility.

Example 2 Patterning of Cells on a Support

[0278] In this example, macro-wells of a stencil which is engaged with athe first layer 150 and support are filled with PBS and a vacuum isapplied for two minutes to remove air bubbles. The support may then betreated with fibronectin (50 mg/ml) or other extracellular matrixprotein for 30 minutes, followed by washing twice with PBS. Afteraspirating PBS, cells may then be plated in freshly warmed medium at adensity of 5-25×10³ cells/cm² (=1-4×10⁴ cells per macro-well of a24-well plate 100, in a volume of 300 ml per macro-well; or 5-25×10⁴cells per 35 mm dish in a volume of 2 ml). The cells deposit through themicro-orifices of the first layer, and attach to the support.

[0279] After the cells have attached to the support (30 minutes-2hours), the cell culture medium in each macro-well is replaced withfresh medium. Cells are left to spread in a 37° C. incubator for twohours to overnight. The cells are washed with PBS and fresh mediumcontaining the treatment of interest is added to the wells. Thestencil/first layer is then removed and the effects of the test compoundon-cell motility, cell shape or viability are observed.

Example 3 Image Acquisition

[0280] Imaging is performed using an inverted microscope equipped withthe following: epifluorescence, motorized and programmable stage,autofocus mechanism, and CCD camera. Two to three randomly selectedareas per macro-well are imaged. The stage translated from onemacro-well to another, and images were focused using automatic focus (Zaxis). Images were captured in either phase contrast or epifluorescence.

[0281] Acquired images shared a common file name, but different suffixcorresponding to the macro-well number and position. For example, anexperiment called TEST with 24 wells generated TEST01-TEST24 when oneimage per macro-well was taken. Images are generated prior toapplication of a test compound or other external stimulus, and atvarious times after treatment.

Example 4 Data Analysis

[0282] Automated data analysis was performed using software thatprocessed information in the following order: a) recall of files inconsecutive order; b) identify cells (using various methods such asthresholding, erosion, and gradient contrasting; c) define cells in acluster using a clustering algorithm; d) measure relevant parameters.Some of the relevant parameters are based on cellular clusters ormicro-regions: average values of perimeter, diameter, surface area,percentage of cell coverage per unit area, perimeter to surface arearatio, and other parameters. The data analysis is capable of correlatingany or all these parameters with cell motility. The final data set maybe based on normalized average of multiple parameters or one specificparameter based on biological observation.

Example 5 3T3/TAXOL

[0283] Macro-wells orifices of a stencil engaged with a support werefilled with PBS and a vacuum was applied for two minutes to remove airbubbles. NIH-3T3 fibroblast cells (prelabeled with green cell tracker,CMFDA, Molecular-Probes) were collected in DMEM/10%—bovine calf serumand plated in the macro-wells at a concentration of 2×10⁴ cells/cm2.After one hour, unadhered cells were washed off with fresh medium. Afteran overnight incubation, fresh medium containing increasing dosages ofpaclitaxel (Sigma, 0.1-10 mg/ml) was added to the wells and the stencilwas peeled off. Control cells were left untreated. Images of migratingcells were taken at time points, from 0-24 h.

Example 6 Farnesyl Transferase Inhibition in MS1 and SVR

[0284] The qualitative cell migration assay plates of the presentinvention are useful in the study of biological pathways, such as theRAS pathway, for example. The assays allow for the study of variousmetabolic pathways and allows for analysis of the effect(s) of agents orbiological entities such as inhibitors of cell migration and/or motilityon cell motility or cell shape. RAS (a guanine nucleotide bindingprotein) plays a pivotal role in the control of both normal andtransformed cell growth. Following stimulation by various growth factorsand cytokines, RAS activates several downstream effectors, leading togene transcription and proliferation. In many cancers, including 90% and50% of pancreatic and colon cancers respectively, ras gene mutationsproduce a mutated RAS that remains locked in an active state, therebyrelaying uncontrolled proliferative signals. Much is known about the RASpathway including strategies to inhibit it. For example, FarnesylTransferase inhibitors inhibit RAS targeting the cell membrane sinceFarnesyl Transferase is believed to assist RAS in membrane localization.Additionally, it is believed that downstream effectors, P13-K and MAPK,can be inhibited, thus in turn inhibit the effect of RAS.

[0285] Using standard protocols, MS1 (T antigen-immortalized endothelialcells, ATCC) and SVR (H-ras-overexpressing derivative of MS1, ATCC) wereplated into macrowells at densities of 12×10³ and 6×10³ cells/cm²,respectively, in DMEM/5% fetal bovine serum. Unattached cells werewashed off after 1 hour, and the cells were replenished with freshmedia. To the media was also added farnesyl transferase inhibitor(FTI-277, Calbiochem) to concentration of 10 mm. Cells were culturedovernight under fresh media in an incubator at 37° C. and 5% CO₂. At thestart of experiment, the stencil/first layer was removed after first onthe support to allow cell migration. At different time points (time zeroand time four hours) images were taken and analyzed for effects ofFTI-277 on cell motility. FIG. 21 contains the pictorial results of anassay showing farnesyl transferase inhibition in MS 1 and SVR cells. Thecontrol cells are shown to have migrated further away from theiroriginal starting positions than the cells treated with FTI. FIG. 22graphically depicts the results of the same assay as shown in FIG. 21.

[0286]FIG. 23 presents the results of an assay where the effects ofseveral inhibitors in the RAS pathway were measured. The graph revealsthat the various inhibitors (P13-K, MAPK, and a mixture of both) show aneffect on the diameter of the cell islands. Measurements were taken at0, 2, 4, 6 and 8 hour increments. Over time, the control cells showed alarger increase in diameter over the cells treated with the inhibitors.The graph reveals that the combination therapy had a greater effect oncell motility (the diameter increased less as the cells moved less).

Example 7 Inhibition of Cell Motility of Renal Cells via MatrixMetalloproteinase Inhibition

[0287] Two renal cell lines were used to study the effect of matrixmetalloproteinase (MMP) inhibition on cell motility. Standard protocolswere used to plate 100 769-P cells (renal carcinoma, purchased fromATCC) and HK-2 (proximal tubule cells from human kidney, from ATCC) inqualitative cell migration assay plate.

[0288] After allowing the cells to attach and spread for 8 hours, thestencil and the first layer were removed and MMP inhibitor (GM6001,Calbiochem) was added at various concentrations. The following datarepresents that MMP inhibition reduces cell motility of 769-P, but hasno effect on the HK-2 cell line. Comparison of a qualitative cellmigration assay plate to a conventional motility assay using BectonDickinsions' transwell (6 well, 8 micron pores) showed that thequalitative cell migration assay plate data correspond to the transwelldata. See FIG. 23, which demonstrates that data from CMA showed moresensitive determination of cell motility.

Example 8 Microtubule Experiments

[0289] Microtubule formation is necessary for cell movement and celldivision. Common cancer drugs such as colchicine, nocodazole,vinblastine and paclitaxel are known to effect cell movement andmigration by acting on the cell's microtubules. Colchicine, nocodazoleand vinblastine disrupt the cell's normal tubulin equilibrium. Thesedrugs “tie up” the tubulin that is present in the cell cytoplasm. Thiscauses the tubulin that is present in the microtubules to disassembleand reenter the cytoplasm to reestablish equilibrium. These drugs alsodisrupt microtubule formation by interacting with binding sites on themicrotubules, causing them to break up.

[0290] A first layer 150 having multiple orifices was applied to asupport 140. The orifices (100 mM diameter holes, separated by 500 mM)were rendered inert to the adsorption of proteins and the adhesion ofcells using the standard procedures described in earlier disclosures:silanes terminated with ethylene glycol groups were reacted covalentlywith the surface of the PDMS devices. The stencil was washed three timeswith PBS, and vacuum was applied for two minutes to remove air bubbles.Human microvasular endothelial cells from lung (HMVEC-L,Clonetics/Biowhittaker), were seeded into the macro-wells of the stencilat a density of 5×10³ cells/cm2 into the dishes in growth medium (EGM,Clonetics/Biowhittaker), and washed with fresh medium after an initialattachment period of 30 minutes to one hour. After an overnightincubation in the membranes, cells were treated with the followingmicrotubule-disrupting agents: nocodazole (10 mg/ml), colchicine (10mg/ml), vinblastine (10 mg/ml), or paclitaxel (10 mg/ml). Control cellswere left untreated. Two different experiments were then performed.

[0291] In the first experiment, cells were treated with the compoundswhile maintained within the macro-wells of the stencil. After two hoursof treatment, the cells were imaged, the stencil was peeled off, and thecells were fixed with cold methanol (−20° C.) for ten minutes and washedthree times with PBS. Immunofluorescence staining was performed using amonoclonal antibody to alpha-tubulin (1:100 dilution, DM1a, Sigma),followed by a FITC-conjugated goat anti-mouse antibody (25 mg/ml,Rockland Immunochemicals) and DAPI (3 mg/ml, Sigma) to -stain thenucleus. Stained cells were mounted under a glass coverslipwith-Fluoromount G (Southern Biotechnology Associates) and imaged in aZeiss fluorescence microscope.

[0292] In the second experiment, the stencil was peeled at the time ofcompound addition. After two hours of treatment, one set of samples wasfixed and stained as described above. Another set of samples was left inthe treatment compound and imaged over time, to monitor cell motility.Images were taken at 0, 2, 4, 8, and 24 hours. Cell motility wasdetermined by taking the average diameter of the micro-regions, usingImageProPlus imaging software.

[0293] While several embodiments have been described above it should beunderstood that these are only illustrative and that others also withinthe spirit and scope of the present invention are also plausible.

We claim:
 1. An assay for monitoring cell movement comprising the stepsof: positioning a first layer to be in fluid-tight contact with asupport, the first layer having an upper surface and defining a patternof micro-orifices, each micro-orifice of the pattern of micro-orificeshaving walls and defining a micro-region on the support when the firstlayer is placed in fluid-tight contact with the support such that thewalls of said each micro-orifice and the micro-region on the supporttogether define a micro-well; positioning a second layer to be influid-tight contact with an upper surface of the first layer, the secondlayer defining a-pattern of macro-orifices, each macro-orifice of thepattern of macro-orifices having walls and defining a macro-region whenthe first layer is placed in fluid-tight contact with the support andthe second layer is placed in fluid-tight contact with the first layersuch that the walls of the macro-orifice and the macro-region togetherdefine a macro-well; each macro-region encompassing at least onemicro-region; immobilizing at least one cell of a plurality of cells ineach respective micro-region on the support so as to situate the atleast one cell within a corresponding micro-well, the cells therebybeing arrayed on the support in a pattern that corresponds to thepattern of the micro-orifices; allowing the cells to grow to confluencywithin the micro-regions; providing at least one of a plurality of testagents to at least one macro-well and allowing said test agent tocontact confluent cells; removing said first and second layer;monitoring cells for movement or lack of movement away from saidmicro-regions; and correlating cellular movement or lack of movementaway from said micro-regions with effect of said test agent on cellularmovement.
 2. The assay of claim 1, wherein each macro-region encompassesa plurality of micro-regions.
 3. The assay of claim 1, wherein the stepof immobilizing at least one cell comprises immobilizing a single cellin each micro-region.
 4. The assay of claim 1, wherein the step ofimmobilizing at least one cell comprises immobilizing a different typeof cell in each micro-region.
 5. The assay of claim 1, wherein the stepof immobilizing at least one cell includes immobilizing at least twodifferent types of cell in a single micro-region.
 6. The assay of claim1 wherein the step of providing at least one test agent of a pluralityof test agents comprises providing a different test agent in eachmacro-well.
 7. The assay of claim 1, further comprising the step ofapplying a coating to an upper surface of the support before positioningsaid first layer.
 8. The assay of claim 7, wherein the step of applyinga coating to an upper surface of the support before positioning saidfirst layer includes applying to the upper surface of the support acoating that is made of a material selected from the group consisting ofproteins, protein fragments, peptides, small molecules, lipid bilayers,metals, self-assembled monolayers, cells, extracellular matrix proteins,hydrogels, and matrigel.
 9. An assay for monitoring cell movementcomprising the steps of: positioning a first layer to be in fluid-tightcontact with a support, the first layer having an upper surface anddefining a pattern of micro-orifices, each micro-orifice of the patternof micro-orifices having walls and defining a micro-region on thesupport when the first layer is placed in fluid-tight contact with thesupport such that the walls of said each micro-orifice and themicro-region on the support together define a micro-well; immobilizingat least one cell of a plurality of cells in each respectivemicro-region on the support so as to situate the at least one cellwithin a corresponding micro-well, the cells thereby being arrayed onthe support in a pattern that corresponds to the pattern of themicro-orifices; allowing the cells to grow to confluency within themicro-regions; removing the first layer from the support after the stepof immobilizing; positioning a second layer to be in fluid-tight contactwith the support, the second layer defining a pattern of macro-orifices,each macro-orifice of the pattern of macro-orifices having walls anddefining a macro-region when the second layer is placed in fluid-tightcontact with the support such that the walls of the macro-orifice andthe macro-region together define a macro-well; each macro-regionencompassing at least one micro-region; providing at least one of aplurality of test agents to at least one macro-well and allowing. saidtest agent to contact confluent cells; removing said second layer;monitoring cells for movement or lack of movement away from saidmicro-regions; correlating cellular movement or lack of movement awayfrom said micro-regions with effect of said test agent on cellularmovement.
 10. The assay of claim 9, wherein each macro-regionencompasses a plurality of micro-regions.
 11. The assay of claim 9,wherein the step of immobilizing at least one cell comprisesimmobilizing a single cell in each micro-region.
 12. The assay of claim9, wherein the step of immobilizing at least one cell comprisesimmobilizing a different type of cell in each micro-region.
 13. Theassay of claim 9, wherein the step of immobilizing at least one cellincludes immobilizing at least two different types of cell in a singlemicro-region.
 14. The assay of claim 1 wherein the step of providing atleast one test agent of a plurality of test agents comprises providing adifferent test agent in each macro-well.
 15. The assay of claim 9,further comprising the step of applying a coating to an upper surface ofthe support before positioning said first layer.
 16. The assay of claim15, wherein the step of applying a coating to an upper surface of thesupport before positioning said first layer includes applying to theupper surface of the support a coating that is made of a materialselected from the group consisting of proteins, protein fragments,peptides, small molecules, lipid bilayers, metals,self-assembled-monolayers, cells, extracellular matrix proteins,hydrogels, and matrigel.
 17. An assay for monitoring cell movementcomprising the steps of: positioning a first layer to be in fluid-tightcontact with a support, the first layer having an upper surface anddefining a pattern of micro-orifices, each micro-orifice of the patternof micro-orifices having walls and defining a micro-region on thesupport when the first layer is placed in fluid-tight contact with thesupport such that the walls of said each micro-orifice and themicro-region on the support together define a micro-well; positioning asecond layer to be in fluid-tight contact with the support, the secondlayer comprising a plurality of rings, the rings defining a pattern ofrespective macro-orifices, each ring having walls and defining amacro-region when the second layer is placed in fluid-tight contact withthe support such that the walls of the ring and the macro-regiontogether define a macro-well; each macro-region encompassing at leastone micro-region; immobilizing at least one cell of a plurality of cellsin each respective micro-region on the support so as to situate the atleast one cell within a corresponding micro-well, the cells therebybeing arrayed on the support in a pattern that corresponds to thepattern of the micro-orifices. allowing the cells to grow to confluencywithin the micro-regions; providing at least one of a plurality of testagents to at least one macro-well and allowing said test agent tocontact confluent cells; removing said first and second layer;monitoring cells for movement or lack of movement away from saidmicro-regions; and correlating cellular movement or lack of movementaway from said micro-regions with effect of said test agent on cellularmovement.
 18. The assay of claim 17, wherein each macro-regionencompasses a plurality of micro-regions.
 19. The assay of claim 17,wherein the step of immobilizing at least one cell comprisesimmobilizing a single cell in each micro-region.
 20. The assay of claim17, wherein the step of immobilizing at least one cell comprisesimmobilizing a different type of cell in each micro-region.
 21. Theassay of claim 17, wherein the step of immobilizing at least one cellincludes immobilizing at least two different types of cell in a singlemicro-region.
 22. The assay of claim 17 wherein the step of providing atleast one test agent of a plurality of test agents comprises providing adifferent test agent in each macro-well.
 23. The assay of claim 17,further comprising the step of applying a coating to an upper surface ofthe support before positioning said first layer.
 24. The assay of claim23, wherein the step of applying a coating to an upper surface of thesupport before positioning said first layer includes applying to theupper surface of the support a coating that is made of a materialselected from the group consisting of proteins, protein fragments,peptides, small molecules, lipid bilayers, metals, self-assembledmonolayers, cells, extracellular matrix proteins, hydrogels, andmatrigel.
 25. An assay for monitoring cell movement comprising the stepsof: positioning a layer to be in fluid-tight contact with the support,the layer defining a pattern of macro-orifices, each macro-orifice ofthe pattern of macro-orifices having walls and defining a macro-regionwhen the layer is placed in fluid-tight contact with the support suchthat the walls of the macro-orifice and the macro-region together definea macro-well; inserting each plug of a set of plugs in a respectivemacro-well, each of the plugs comprising a lower membrane placed influid-tight contact with the support when the layer is placed influid-tight contact with the support and the plug is received in acorresponding macro-well defined by the layer and the support, the lowermembrane further defining a pattern of micro-orifices, wherein eachmicro-orifice has walls and defines a micro-region on the support whenthe plug is in fluid-tight contact with the support such that the wallsof the micro-orifice and the micro-region together define a micro-well;immobilizing a cell in at least one micro-region on the support so as tobe situated within the micro-well, such that the cell is arrayed on thesupport in a pattern that corresponds to the first pattern ofmicro-orifices; allowing the cells to grow to confluency within themicro-regions; providing at least one of a plurality of test agents toat least one macro-well and allowing said test agent to contactconfluent cells; removing said layer containing said plugs; monitoringcells for movement or lack of movement away from said micro-regions; andcorrelating cellular movement or lack of movement away from saidmicro-regions with effect of said test agent on cellular movement. 26.The assay of claim 25, wherein the step of immobilizing a cell comprisesimmobilizing a different type of cell in each micro-region.
 27. Theassay of claim 25, wherein the step of immobilizing a cell includesimmobilizing at least two different types of cells in a singlemicro-region.
 28. The assay of claim 25, wherein the step of providingat least one test agent of a plurality of test agents comprisesproviding a different test agent in each macro-well.
 29. The assay ofclaim 25, further comprising the step of applying a coating to an uppersurface of the support before positioning said first layer.
 30. Theassay of claim 29, wherein the step of applying a coating to an uppersurface of the support before positioning said first layer includesapplying to the upper surface of the support a coating that is made of amaterial selected from the group consisting of proteins, proteinfragments, peptides, small molecules, lipid bilayers, metals,self-assembled monolayers, cells, extracellular matrix proteins,hydrogels, and matrigel.